LOCUS       sequence-426253-        6312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for M-NylC gene, codon optimized for expression
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6312)
  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 6312)
  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: M-NylC_pET28a
FEATURES             Location/Qualifiers
     source          1..6312
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1027..1044
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1097..1115)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1111..1158
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1195..1650
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1282..1301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1492..1513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1742..2557)
                     /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(1742..2557)
                     /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     2465..2484
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2549..2568
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2679..3267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3168..3187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3421..3438
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3453..3592
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3518..3537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3678..3700
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3694..3885)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4694..5776)
                     /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     5738..5757
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5777..5854)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6060..6079
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6163..6182
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6163..6181
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6182..6206
                     /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             6221..6243
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6262..6279
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6289..6306
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 gtgaccgaca cccatcgact cgcgccggtt ctcgccgccg gaccccgtcg ctcaaacggt
       61 gattatgctt tgtcaccagt tcacggtacc gatagaggcc aagtcgagta cgacttccca
      121 ggtgtcctgc tcggtactgc agaatatgca gagggcccga cgggggccac agtgttgagt
      181 attccggcag gagcgcgtac attcactgat cgccgcgggg gtgcggtggg cgcatcaggg
      241 ttgtatggct ataatcatgc catatgtctc gcaggcggtt ccgtttatgg cctgagcgca
      301 gttgccggtg ttagtgaggc tttatttgag agagcggagc atagatctgg ctttgaccag
      361 ctgcagttag tgagtggtgc tattatttac gactattcag tgcgagaaaa cagcgttttt
      421 ccagataccg ccctgggtaa agccgccctt caagccgcgc gtcaaggcgt cgttgaagta
      481 ggtcgcgttg gaggtggagc gagcgcatct tccggtaaag tcgatccggc aagagttgaa
      541 tttactggcc aaggcgtagc attccggcaa gtcggtgatg tcaaagtgtt agtcgtaaca
      601 gtactgaacc cgtatggtgt gatattggac agaagtggtc gtatcatcag aggcaattac
      661 gacgcgagta ccggtgaacg tcgccaccca gcccttgatt atgaagaagc cattggcgag
      721 tctcgtcttg ttgattctat gtcgggcaat accactatta cggccgtcgt caccaacgtc
      781 caattgacag ataccgaact gaagcaattt ggcttacagg tgcattcttc tatgcaccgc
      841 ggcatacagc cattccatac gcccctggac ggagacactc tttttgccct tactactgat
      901 gaagtggcgc tgccggaaaa tccgtcaaca tccagaggcc gtctctcgtt gaatagcacg
      961 gccgtcgcaa ccctggcggg ggaaacggct tgggatgcga tcttatgtgc ggctggttga
     1021 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1081 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1141 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1201 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1261 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1321 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1381 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1441 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1501 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1561 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1621 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1681 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     1741 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1801 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1861 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1921 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1981 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     2041 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2101 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2161 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2221 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     2281 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2341 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2401 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     2461 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2521 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     2581 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2641 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2701 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2761 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2821 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2881 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2941 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3001 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3061 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3121 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3181 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3241 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3301 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3361 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3421 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3481 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3541 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     3601 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3661 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3721 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     3781 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     3841 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     3901 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     3961 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4021 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4081 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4141 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4201 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     4261 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     4321 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     4381 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     4441 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     4501 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     4561 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     4621 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     4681 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4741 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     4801 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     4861 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     4921 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     4981 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     5041 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     5101 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     5161 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     5221 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     5281 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     5341 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     5401 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     5461 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     5521 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     5581 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     5641 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     5701 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     5761 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     5821 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     5881 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     5941 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     6001 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     6061 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     6121 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     6181 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     6241 agatatacca tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc
     6301 ggcagccata tg
//
