LOCUS       sequence-425835-        5888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for AoC gene, codon optimized for expression in
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5888)
  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 5888)
  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: AoC_pET29b
FEATURES             Location/Qualifiers
     source          1..5888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     sig_peptide     1..60
                     /codon_start=1
                     /product="signal sequence of E. coli alkaline phosphatase 
                     (Inouye et al., 1982)"
                     /label=phoA signal sequence
                     /translation="KQSTIALALLPLLFTPVTKA"
     CDS             658..675
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(728..746)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      742..789
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      826..1281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(913..932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1123..1144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1373..2188)
                     /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(1373..2188)
                     /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     2096..2115
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2180..2199
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2310..2898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2799..2818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3052..3069
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3084..3223
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3149..3168)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3309..3331
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3325..3516)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4325..5407)
                     /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     5369..5388
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5408..5485)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5691..5710
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5798..5817
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5798..5816
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5817..5841
                     /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             5856..5878
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 aaacagtcca ctatcgcatt ggccctgtta ccactgctgt ttaccccggt gacgaaagcg
       61 agccctgtag atttacagga tcgccagctg actggcgggg acgaattacg cgatggtccg
      121 tgcaagccga tcacgttcat cttcgcacgc gctagcaccg aaccgggctt gctgggcatc
      181 tctaccggcc cagccgtttg caatcgcctg aaactggcgc gcagtggaga tgttgcgtgt
      241 caaggcgtgg gcccgcgcta taccgcggat ctgccctcta acgccctgcc ggaggggacc
      301 agtcaggcgg cgatcgcaga ggcgcagggc ctctttgaac aggccgtcag taagtgcccc
      361 gatacgcaga tcgtggcggg tgggtatagc cagggcaccg cggtaatgaa tggtgccatt
      421 aaacggctgt cagcagatgt gcaggacaaa attaaagggg tggttttatt tgggtatacg
      481 cgcaacgcgc aggaacgtgg ccagattgca aatttcccga aggataaggt aaaagtttat
      541 tgcgcggttg gtgacctggt atgtctgggc acgctgatcg tcgcgccccc gcacttctca
      601 tatctgagtg acacgggtga tgcgtccgat tttctgttat ctcaattagg tctcgagcac
      661 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      721 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      781 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      841 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      901 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      961 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1021 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1081 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1141 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1201 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1261 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1321 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1381 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1441 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1501 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1561 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1621 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1681 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1741 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1801 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1861 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1921 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     1981 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2041 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2101 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2161 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2221 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2281 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2341 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2401 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2461 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2521 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2581 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2641 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2701 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2761 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2821 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2881 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2941 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3001 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3061 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3121 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3181 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3241 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3301 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3361 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3421 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3481 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3541 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3601 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     3661 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     3721 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     3781 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     3841 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     3901 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     3961 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4021 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4081 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4141 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4201 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4261 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4321 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4381 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4441 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4501 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4561 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     4621 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4681 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4741 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4801 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4861 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     4921 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     4981 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5041 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5101 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5161 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5221 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5281 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5341 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5401 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5461 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5521 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5581 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     5641 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     5701 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     5761 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     5821 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     5881 tacatatg
//
