LOCUS       sequence-426312-        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for Nc-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 6324)
  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 6324)
  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: Nc-NylC_pET28a
FEATURES             Location/Qualifiers
     source          1..6324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1039..1056
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1109..1127)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1123..1170
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1207..1662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1294..1313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1504..1525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1754..2569)
                     /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(1754..2569)
                     /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     2477..2496
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2561..2580
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2691..3279
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3180..3199
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3433..3450
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3465..3604
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3530..3549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3690..3712
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3706..3897)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4706..5788)
                     /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     5750..5769
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5789..5866)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6072..6091
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6175..6194
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6175..6193
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6194..6218
                     /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             6233..6255
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6274..6291
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6301..6318
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 aatgaacgta cagccgcaga cgcggctcat ggcgatcccg ctcgcggtga cgatgtaaga
       61 gcagtcgcga cgtcgcggta cgctgaaggc gcaccaggta acgacgatgt ccgaccagta
      121 ccggctccat ctcccggtcg tgcggatgtg gcgtttgata tccccggggt cagcgtggga
      181 acagcagaat acaccgaagg tccgacgggg tgtacagtta tccggtttga cggggctgcc
      241 cgaactgcga tagacgctcg gggtggtgca gttggcttgt ccggtggcta tgaagttcat
      301 caggccatag tatttgcagg ggggtcggtt tatggtttag aagccgctgc tggtgtcggt
      361 acgggcctgt tacgagaaca gggtaaccgg acccattggg ccgcattgca gcatgtttcg
      421 ggagccgtga tatatgattt cagtgctcgt gacacggcaa tagcgcctga cgcagccctc
      481 ggtcgggcgg ccttacgcgc agcgcgtccg ggccggtttc ccgtaggacg ctgtggcgcc
      541 ggccgctcag cttctgttgg taaaatagat ttttctcgcg ccgaatttgc gggtcaaggt
      601 gcggcgtacc gggcttttgg cccagtaaag gtgttagttg ccactgtcgt aaatgccgtc
      661 ggtgttgtag tcgatcgtga cggtcgtgtt gtcagaggga atgtgcatcc agatggccgt
      721 cgtcgccgtc ccgcggatga cgtcgcggct ggactgggtt caggtgatgg ccctgcgccg
      781 ttagccggaa ataccacatt aacggtgctg gtgactaatg ctcgcctgcc agaacgttca
      841 cttcgtcagc tcggcacaca agtgcacagc tcgatgcatc gtgcgatcca gccgtttcac
      901 acagaaatcg acggcgatat cttatttacg gtaacaacgg atgaagtcga cttggaaggc
      961 accacggcca ccggtttagg tgcgctggcc agtgaagtgg cctgggatgc agtgctctct
     1021 gcggccgagt gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1081 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1141 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1201 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1261 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1321 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1381 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1441 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1501 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1561 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1621 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1681 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1741 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     1801 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     1861 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     1921 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     1981 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     2041 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     2101 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     2161 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     2221 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     2281 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     2341 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2401 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     2461 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     2521 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     2581 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     2641 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2701 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2761 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2821 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2881 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2941 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3001 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3061 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3121 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3181 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3241 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3301 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3361 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3421 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3481 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3541 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3601 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3661 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3721 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3781 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3841 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3901 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3961 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4021 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4081 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4141 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4201 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4261 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4321 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4381 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4441 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4501 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4561 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4621 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4681 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4741 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4801 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4861 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4921 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4981 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5041 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5101 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5161 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5221 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5281 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5341 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5401 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5461 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5521 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5581 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5641 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5701 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5761 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5821 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5881 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5941 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6001 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6061 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6121 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     6181 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     6241 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc
     6301 ctggtgccgc gcggcagcca tatg
//
