LOCUS       pET28a-RF1              6326 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Nucleus PURE: pET28a-RF1 in pET28a protein expression plasmid. See
            https://nucleus.bnext.bio/ for more details..
ACCESSION   pET28a-RF1
VERSION     pET28a-RF1
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..4728
                     /codon_start=1
                     /standard_name="RF1"
                     /label="RF1"
                     /translation="MKPSIVAKLEALHERHEEVQALLGDAQTIADQERFRALSREYAQL
                     SDVSRCFTDWQQVQEDIETAQMMLDDPEMREMAQDELREAKEKSEQLEQQLQVLLLPKD
                     PDDERNAFLEVRAGTGGDEAALFAGDLFRMYSRYAEARRWRVEIMSASEGEHGGYKEII
                     AKISGDGVYGRLKFESGGHRVQRVPATESQGRIHTSACTVAVMPELPDAELPDINPADL
                     RIDTFRSSGAGGQHVNTTDSAIRITHLPTGIVVECQDERSQHKNKAKALSVLGARIHAA
                     EMAKRQQAEASTRRNLLGSGDRSDRNRTYNFPQGRVTDHRINLTLYRLDEVMEGKLDML
                     IEPIIQEHQADQLAALSEQE"
     CDS             4729..4743
                     /codon_start=1
                     /label="Linker"
                     /translation="GGGGS"
     CDS             4744..4761
                     /codon_start=1
                     /label="His6"
                     /translation="HHHHHH"
     terminator      4835..4882
                     /label="T7 terminator"
                     /note="Color: #ffffff NCBI Feature Key: terminator
                     Transferred From: T7_terminator Transferred Similarity:
                     100.00%"
     rep_origin      4919..5374
                     /direction=RIGHT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5466..6281)
                     /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 gaaaccgagc
     3661 atcgttgcaa agttagaagc acttcacgaa cgtcacgaag aagtgcaggc tttactgggc
     3721 gacgctcaga ctattgcgga tcaagaacgc tttcgcgcat tatcacgtga gtacgctcag
     3781 ctgagtgatg ttagccgttg cttcacggat tggcaacagg tgcaagaaga tattgaaact
     3841 gcccaaatga tgctggacga tccagaaatg cgcgagatgg cccaagacga actgcgggag
     3901 gccaaagaaa agtcggaaca gttagagcag cagttgcagg tcttgctgct gccgaaggac
     3961 ccggacgatg agcggaacgc gtttctggag gtgcgtgctg gcacgggcgg tgatgaagct
     4021 gcgctgtttg ccggcgacct gttccgtatg tacagtcgtt atgcggaagc ccgtcgctgg
     4081 cgggtcgaga ttatgagcgc gtcagaaggt gagcatggcg gctacaaaga aatcattgca
     4141 aaaatcagtg gcgacggcgt ttacgggcgc ttgaaatttg agtctggggg tcaccgggtg
     4201 cagcgggtac ccgctaccga atcacagggt cgtattcaca cgtccgcgtg taccgtggca
     4261 gtgatgcccg agttgccgga cgcggaactt ccagatatta accccgccga tcttcgcatt
     4321 gatacctttc gctctagcgg ggcaggggga cagcatgtta ataccaccga ctcagccatt
     4381 cgtattactc acctccctac gggtatcgtg gtggagtgcc aagacgaacg ttcacagcac
     4441 aagaacaaag cgaaagcgct gtcggtgctg ggggctcgta ttcatgcggc ggagatggcg
     4501 aaacgccagc aagctgaagc atcaactcgg cgtaacctgc tgggctccgg agatcggtcg
     4561 gatcgtaacc gtacatataa ttttcctcag gggcgtgtga ccgatcaccg cattaacttg
     4621 accctttatc gcttggatga ggtgatggag ggaaaattgg atatgttaat cgagccgatt
     4681 attcaggaac accaagcaga tcaactggcc gcattgtccg aacaggaggg aggcggtgga
     4741 tctcaccatc accaccatca ttgaagagct tgatccggct gctaacaaag cccgaaagga
     4801 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4861 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     4921 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4981 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5041 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5101 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     5161 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5221 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5281 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5341 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     5401 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     5461 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     5521 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     5581 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     5641 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     5701 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     5761 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     5821 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     5881 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     5941 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     6001 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     6061 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     6121 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     6181 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     6241 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     6301 tttatgtaag cagacagttt tattgt
//
