LOCUS       pET28a-RF2              6344 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Nucleus PURE: pET28a-RF2 in pET28a protein expression plasmid. See
            https://nucleus.bnext.bio/ for more details..
ACCESSION   pET28a-RF2
VERSION     pET28a-RF2
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..4779
                     /codon_start=1
                     /standard_name="RF2"
                     /label="RF2"
                     /translation="MFEINPVNNRIQDLTERSDVLRGYLDYDAKKERLEEVNAELEQPD
                     VWNEPERAQALGKERSSLEAVVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELD
                     ALEEKLAQLEFRRMFSGEYDSADCYLDIQAGSGGTEAQDWASMLERMYLRWAESRGFKT
                     EIIEESEGEVAGIKSVTIKISGDYAYGWLRTETGVHRLVRKSPFDSGGRRHTSFSSAFV
                     YPEVDDDIDIEINPADLRIDVYRTSGAGGQHVNRTESAVRITHIPTGIVTQCQNDRSQH
                     KNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWGSQIRSYVLDDSRIKDLRTGV
                     ETRNTQAVLDGSLDQFIEASLKAGL"
     terminator      4853..4900
                     /label="T7 terminator"
                     /note="Color: #ffffff NCBI Feature Key: terminator
                     Transferred From: T7_terminator Transferred Similarity:
                     100.00%"
     rep_origin      4937..5392
                     /direction=RIGHT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5484..6299)
                     /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 atctatgttt gaaattaatc ctgtgaataa ccgtatccag
     3721 gatctgacgg aacggtcaga tgtgttgcgt gggtacttag attacgatgc aaagaaggag
     3781 cgcttagagg aggttaatgc cgaacttgaa cagcctgatg tgtggaatga gccagagcgc
     3841 gcgcaggcac tgggtaagga gcgttcgtct ttagaggcgg tggtggatac cctggatcag
     3901 atgaaacagg gtttagaaga tgttagtgga ctgctggagc tggcagtgga ggccgatgat
     3961 gaagaaactt tcaacgaagc agttgccgaa ctggatgctc tggaggaaaa actggcacag
     4021 ttagagttcc ggcgcatgtt tagtggtgag tacgatagtg cggattgcta tctggatatt
     4081 caggccggta gtggcgggac agaagcccag gattgggcgt cgatgctgga acgtatgtat
     4141 ctgcgctggg ccgaatcccg tggctttaaa accgaaatta tcgaggaatc ggagggtgaa
     4201 gtagctggta tcaagtcagt caccattaag atttcggggg attacgcata cggctggttg
     4261 cgtaccgaaa ccggcgttca ccgtctggtt cgcaaatcgc catttgacag cggcggtcgg
     4321 cgtcacacct ccttcagctc cgcatttgtg tacccggaag ttgatgatga tattgatatc
     4381 gagatcaatc ccgcggatct tcgtatcgac gtgtatcgca cctctggtgc gggcggtcag
     4441 catgtcaacc ggaccgaaag tgccgtgcgc atcacacata tcccgactgg tatcgtgacg
     4501 cagtgccaga atgatcggag ccagcacaaa aacaaagatc aggcaatgaa acagatgaaa
     4561 gccaaattgt acgaattgga gatgcagaaa aaaaatgcag aaaaacaggc aatggaagat
     4621 aacaagtctg atattgggtg gggctcgcaa atccgcagct atgttctgga tgatagccgc
     4681 atcaaagacc ttcgtactgg cgttgaaacg cgtaacactc aggctgtcct tgatggcagc
     4741 cttgatcagt tcattgaggc gagtcttaaa gcgggcctgt gaagagcttg atccggctgc
     4801 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     4861 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     4921 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5341 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5401 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5461 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     5521 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     5581 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5641 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5701 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     5761 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     5821 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     5881 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     5941 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     6001 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     6061 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     6121 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     6181 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     6241 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     6301 acaccccttg tattactgtt tatgtaagca gacagtttta ttgt
//
