LOCUS       sequence-433775-        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for BcC gene, codon optimized for expression in
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  AUTHORS   Bell EL, Rosetto G, Ingraham MA, Ramirez KJ, Lincoln C, Clarke RW, 
            Gado JE, Lilly JL, Kucharzyk KH, Erickson E, Beckham GT
  TITLE     Natural diversity screening, assay development, and characterization
            of nylon-6 enzymatic depolymerization.
  JOURNAL   Nat Commun. 2024 Feb 9;15(1):1217. doi: 10.1038/s41467-024-45523-5.
  PUBMED    38336849
REFERENCE   2  (bases 1 to 6140)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-02-9"; volume: "15(1):1217. doi"; pages: " 
            10.1038/s41467-024-45523-5"
COMMENT     Sequence Label: BcC _pET29b
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             910..927
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(980..998)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      994..1041
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1078..1533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1165..1184)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1375..1396
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1625..2440)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1625..2440)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2348..2367
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2432..2451
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2562..3150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3051..3070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3304..3321
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3336..3475
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3401..3420)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3561..3583
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3577..3768)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4577..5659)
                     /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"
     primer_bind     5621..5640
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5660..5737)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5943..5962
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6050..6069
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6050..6068
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6069..6093
                     /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             6108..6130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 cggaccagac gactcgcaca atggctgtcc ccagctagag cagcctttac gactgttgcg
       61 cttgccgctg cctgcctcag tgcccctgca attgcggcgg attatgccaa agggcctgcg
      121 cctacagtgg ctcttttaac tgcccctgcc ggtccatatc aaacagccac atataaaatc
      181 gctacggcag atgctaaagc ctttaattat gggggtgcta cggttcatta ccccgtagct
      241 aaagcaccag gtgagacttt tggcgtagtg gttatggtgc caggatttct ggcttttcag
      301 gcagtttatg actggcttgt tagccgggtg gcgtcacatg gctttgtcgt agttaacatg
      361 gacacggtgg gacgcggcga ctatccggac gaacgcgctg aacaagctgc aggcgctctt
      421 cgccacgtgg tgtcccttgc ccagggcggt acggcccctt acgctccggt cgcggacacc
      481 agccgtcggg ctgtcatggg taattcaatg ggcggaggtg ccagtcttag tgctgctgtt
      541 gccgatccaa ccttaaaagc tgtcgtagct ctgcagcctt ggcacacaac caagagtttc
      601 accggtgtag cctcacccac gttagtcgtt gcctgcgaaa aggattccat tgccccgaac
      661 aaatcacatt cggacgtttt ttataatagc ttgtcagctg cgctgccacg tgcagagatc
      721 gagatgcgtg gtgcaggcca tttatgcgcg acgtttcttg caacagccgc acagaaaacg
      781 acaatggcca agagttccgt cgcgtggtta aaacggtttc tcgacaacga catgcggtac
      841 gacgcaatgg ttaaaggcgg catagatcaa ggggaatttt ccagattcgt cgttcagggc
      901 tttctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      961 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1021 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1081 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1141 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1201 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1261 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1321 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1381 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1441 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1501 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1561 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1621 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1681 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1741 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     1801 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     1861 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     1921 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     1981 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2041 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     2101 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     2161 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     2221 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     2281 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     2341 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     2401 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     2461 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     2521 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2581 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2641 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2701 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2761 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2821 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2881 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2941 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3001 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3061 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3181 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3241 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3301 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3361 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3421 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3481 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3541 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3601 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3661 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3721 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3781 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3841 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3901 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     3961 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4021 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4081 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4141 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     4201 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     4261 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4321 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4381 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4441 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4501 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4561 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4621 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4681 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4741 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4801 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     4861 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4921 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4981 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5041 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5101 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5161 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5221 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5281 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5341 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5401 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5461 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5521 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5581 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5641 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5701 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5761 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5821 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5881 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5941 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6001 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt aatacgactc
     6061 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     6121 aagaaggaga tatacatatg
//
