LOCUS       pET28a-AlaRS            7877 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Nucleus PURE: pET28a-AlaRS in pET28a protein expression plasmid. See
            https://nucleus.bnext.bio/ for more details..
ACCESSION   pET28a-AlaRS
VERSION     pET28a-AlaRS
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     rep_origin      77..665
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     promoter        77..105
                     /label="RNAII promoter"
                     /note="Color: #ffffff NCBI Feature Key: promoter NCBI Join
                     Type: join Transferred From: RNAII_promoter Transferred
                     Similarity: 100.00%"
     misc_RNA        113..665
                     /label="RNAII transcript (complementary strand)"
                     /note="RNAII transcript (complementary strand)"
     misc_RNA        complement(116..223)
                     /label="RNAI transcript"
                     /note="RNAI transcript"
     promoter        complement(230..259)
                     /label="RNA I promoter"
                     /note="Color: #ffffff NCBI Feature Key: promoter NCBI Join
                     Type: join Transferred From: RNA_I_promoter Transferred
                     Similarity: 100.00%"
     terminator      669..712
                     /label="rop/bom terminator"
     misc_binding    752..760
                     /label="dnaA binding site"
                     /bound_moiety="dnaA"
     misc_feature    785..848
                     /label="L-strand Y effector site"
                     /note="L-strand Y effector site"
     misc_feature    851..990
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     misc_feature    1031..1187
                     /label="H-strand Y effector site"
                     /note="H-strand Y effector site"
     CDS             complement(1092..1283)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /label="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             1284..1293
     misc_feature    complement(1359..1377)
                     /label="ROP calculated promoter"
     CDS             complement(2092..3174)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label="lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3175..3252)
                     /label="lacI promoter"
                     /note="Color: #ffffff NCBI Feature Key: promoter
                     Transferred From: lacI_promoter Transferred Similarity:
                     100.00%"
     promoter        3561..3579
                     /label="T7_promoter"
     protein_bind    3580..3604
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             3613..3645
                     /label="UTR1"
                     /note="note: UTR1 NCBI Feature Key: misc_feature Color:
                     #cc0000 modified by: Andrey Transferred From: U4m - UTR1
                     Transferred Similarity: 100.00%"
     CDS             3649..3651
                     /codon_start=1
                     /label="Start"
                     /translation="M"
     CDS             3652..3669
                     /codon_start=1
                     /label="His6"
                     /translation="HHHHHH"
     CDS             3670..3684
                     /codon_start=1
                     /label="Linker"
                     /translation="GGGGS"
     CDS             3685..6312
                     /codon_start=1
                     /standard_name="AlaRS"
                     /label="AlaRS"
                     /translation="MSKSTAEIRQAFLDFFHSKGHQVVASSSLVPHNDPTLLFTNAGMN
                     QFKDVFLGLDKRNYSRATTSQRCVRAGGKHNDLENVGYTARHHTFFEMLGNFSFGDYFK
                     HDAIQFAWELLTSEKWFALPKERLWVTVYESDDEAYEIWEKEVGIPRERIIRIGDNKGA
                     PYASDNFWQMGDTGPCGPCTEIFYDHGDHIWGGPPGSPEEDGDRYIEIWNIVFMQFNRQ
                     ADGTMEPLPKPSVDTGMGLERIAAVLQHVNSNYDIDLFRTLIQAVAKVTGATDLSNKSL
                     RVIADHIRSCAFLIADGVMPSNENRGYVLRRIIRRAVRHGNMLGAKETFFYKLVGPLID
                     VMGSAGEDLKRQQAQVEQVLKTEEEQFARTLERGLALLDEELAKLSGDTLDGETAFRLY
                     DTYGFPVDLTADVCRERNIKVDEAGFEAAMEEQRRRAREASGFGADYNAMIRVDSASEF
                     KGYDHLELNGKVTALFVDGKAVDAINAGQEAVVVLDQTPFYAESGGQVGDKGELKGANF
                     SFAVEDTQKYGQAIGHIGKLAAGSLKVGDAVQADVDEARRARIRLNHSATHLMHAALRQ
                     VLGTHVSQKGSLVNDKVLRFDFSHNEAMKPEEIRAVEDLVNTQIRRNLPIETNIMDLEA
                     AKAKGAMALFGEKYDERVRVLSMGDFSTELCGGTHASRTGDIGLFRIISESGTAAGVRR
                     IEAVTGEGAIATVHADSDRLSEVAHLLKGDSNNLADKVRSVLERTRQLEKELQQLKEQA
                     AAQESANLSSKAIDVNGVKLLVSELSGVEPKMLRTMVDDLKNQLGSTIIVLATVVEGKV
                     SLIAGVSKDVTDRVKAGELIGMVAQQVGGKGGGRPDMAQAGGTDAAALPAALASVKGWV
                     SAKLQ"
     terminator      6386..6433
                     /label="T7 terminator"
                     /note="Color: #ffffff NCBI Feature Key: terminator
                     Transferred From: T7_terminator Transferred Similarity:
                     100.00%"
     rep_origin      6470..6925
                     /direction=RIGHT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(7017..7832)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      961 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1021 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1081 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1141 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1201 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1261 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1321 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     1381 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     1441 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     1501 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1561 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1621 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1681 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1741 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1801 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1861 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1921 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1981 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     2041 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     2101 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2161 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     2221 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     2281 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     2341 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     2401 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     2461 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     2521 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     2581 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     2641 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2701 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2761 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2821 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2881 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2941 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     3001 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     3061 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     3121 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     3181 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     3241 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     3301 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     3361 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     3421 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     3481 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     3541 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     3601 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccaat gcaccatcac
     3661 caccatcatg gaggcggtgg atctatgtcg aagtcgaccg ccgagattcg tcaagcgttt
     3721 ctggatttct ttcattcgaa aggccaccag gtggttgctt ctagttcgct ggtgccgcac
     3781 aatgatccga ccctgttgtt cacgaatgca gggatgaatc aatttaaaga tgtgttcctg
     3841 ggcctggata aacgcaatta ttcacgcgcc acaacatctc agcgttgcgt ccgtgccggt
     3901 ggcaaacata acgatctgga aaacgttggc tatactgcgc ggcaccatac atttttcgaa
     3961 atgctgggca acttcagttt tggagactat ttcaagcatg atgcaatcca gtttgcgtgg
     4021 gaattgctga cctctgaaaa atggttcgcc ttgccaaaag agcgtctttg ggtaaccgta
     4081 tatgagtcag acgatgaagc ctatgaaatt tgggaaaaag aggtgggtat cccccgcgaa
     4141 cgcattattc gcattgggga taacaaaggc gccccttacg cgtctgataa cttctggcag
     4201 atgggcgata ccggcccgtg cgggccttgt accgaaattt tttacgacca tggtgaccac
     4261 atctggggag gtccgccggg gtcgccagaa gaagatggcg atcgctatat tgaaatctgg
     4321 aatattgtgt ttatgcagtt caaccgtcag gccgacggca caatggaacc gctgcctaaa
     4381 cccagcgttg atactggtat ggggcttgaa cgtatcgccg ccgtgctgca acatgtcaac
     4441 agcaattatg acatcgatct gtttcgtacg ctgatccagg ctgtggcgaa ggttacagga
     4501 gcgaccgatc tgtctaataa aagtctgcgt gtgattgcgg atcacatccg ctcgtgcgct
     4561 tttctgatcg cggacggtgt gatgccaagt aatgaaaatc gcgggtatgt actgcgccgc
     4621 atcattcgcc gtgccgtacg gcacggaaat atgcttggtg ctaaagagac attcttttat
     4681 aaactggtcg gcccactgat cgatgtgatg ggaagtgcgg gtgaagattt gaaacgtcag
     4741 caggcgcagg ttgaacaagt gttaaaaacc gaagaagaac agtttgcccg caccctggaa
     4801 cgtggcttgg cgcttttgga cgaagaactg gcaaaactgt ctggcgacac cttggacgga
     4861 gagacagcat ttcgcttata tgatacatac ggctttccgg tcgatttgac ggctgatgtt
     4921 tgccgcgaac ggaatattaa ggttgatgaa gccggctttg aggcagccat ggaagaacaa
     4981 cgtcgccggg cgcgcgaagc cagtggcttt ggcgctgatt ataatgccat gattcgcgtt
     5041 gattcagcgt cagaatttaa gggctacgac cacttagaac tgaatggcaa agtgaccgct
     5101 ctgttcgtag atggcaaggc ggtggatgcg atcaatgcgg gtcaagaggc ggttgtggtt
     5161 ttggatcaaa cgccctttta tgccgaatct ggcggacagg tgggtgataa aggcgaactt
     5221 aaaggcgcta atttcagctt cgcggtggag gatactcaaa aatatggcca ggctatcggt
     5281 catatcggta aactggccgc gggttctctg aaggtcggag atgcggtcca ggctgatgtc
     5341 gatgaggcac gtcgtgcgcg cattcgttta aatcattcgg cgacccacct gatgcacgca
     5401 gcgttacgcc aagtgctggg tacccatgtt tcccagaaag gctccctggt gaatgacaaa
     5461 gtcttacgtt tcgatttcag tcacaacgag gccatgaagc cggaagaaat ccgtgcggtg
     5521 gaagatctgg tgaacaccca gatccgccgg aatttgccta tcgaaaccaa tattatggac
     5581 ttagaagccg caaaagcgaa aggagccatg gcgctgtttg gcgaaaaata tgatgaacgt
     5641 gtgcgtgtcc ttagcatggg ggacttcagt acagagttat gcggaggtac ccacgctagt
     5701 cgcacgggtg atattggctt gtttcgtatc atttctgagt ccggtacagc agccggtgtg
     5761 cgccggatcg aagctgttac gggtgagggc gcgatcgcta cggtgcatgc tgacagcgat
     5821 cgcctgagcg aagttgccca tcttttaaaa ggggactcca acaatctggc agataaagtg
     5881 cgttctgtcc tggaacgtac acgtcagttg gaaaaagagt tgcaacagct gaaggagcag
     5941 gcagcagctc aggaaagcgc aaatcttagc tctaaagcca ttgatgtgaa cggggtgaaa
     6001 ttgctggtta gcgaactgag cggagttgaa ccgaagatgc ttcgcaccat ggtggatgac
     6061 ttaaagaacc aattaggatc tacgatcatc gtgctggcga ctgttgtcga aggtaaagta
     6121 tcgttaattg cgggggtgtc gaaagatgtg actgatcgtg taaaagcagg ggaattgatc
     6181 ggtatggtcg cccagcaggt aggtggcaaa ggcggaggac gcccagatat ggcccaagct
     6241 gggggtaccg atgcggcagc actgccggct gcattggcga gcgtaaaggg atgggtttcc
     6301 gctaaattac agtgaagagc ttgatccggc tgctaacaaa gcccgaaagg aagctgagtt
     6361 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     6421 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     6481 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6541 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6601 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6661 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     6721 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     6781 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     6841 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     6901 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6961 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     7021 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     7081 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     7141 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     7201 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     7261 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     7321 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     7381 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     7441 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     7501 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     7561 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     7621 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     7681 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     7741 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     7801 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     7861 gcagacagtt ttattgt
//
