LOCUS       sequence-425829-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for OoH gene, codon optimized for expression in
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  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 6671)
  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: OoH_pET29b
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1441..1458
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1511..1529)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1525..1572
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1609..2064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1696..1715)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1906..1927
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2156..2971)
                     /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(2156..2971)
                     /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     2879..2898
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2963..2982
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3093..3681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3582..3601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3835..3852
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3867..4006
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3932..3951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4092..4114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4108..4299)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5108..6190)
                     /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     6152..6171
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6191..6268)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6474..6493
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6581..6600
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6581..6599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6600..6624
                     /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             6639..6661
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gacgttgccg aatacgcggc tcacgacgcg acaggcctgg cggaattaat ccgagatgga
       61 caggtcagtg cagccgaagt ttggactgca gcggcgacag ctttagatgc ggttgaacca
      121 gaactggctg cggtggcagg ggaacgtttt gataagccgc tggattatga tgaaagtggc
      181 ccttttgctg gagtaccttt tgccctcaaa gatctgattg ctcatgctgg aggtgtcccg
      241 tcgcgcagtg gctcacgtct cttcggagcc ggtgtggcac atccggaaga tacgcactta
      301 gtcgcgcggt tcagacgtgc gggccttgct atcggggcaa ttacccgttc accggagttt
      361 ggttttaata cgaccacgga agctatagcg tatggtggcc cgagccgcaa tccgtgggcc
      421 acggaccgct cgcctggagg atcatcggga gcaagtgcgg cgcttgttgc gtcgggcgca
      481 ttgccaatgg cgcatgcgaa tgatggcggc gggtccattc gcatacctgc tgcagcatgc
      541 ggtgccgtcg ggttgaagcc ttctcgcggc cggacaactc caggcccaga tctggctgac
      601 cctttactgg gccttgtgat cgaatttgct gttacccgca cagtccggga ttgcgcgcgg
      661 ttgctcgatg ccgttcacgg ggcagagcct ggtgatcgtt atcttctccc tgggcctgtt
      721 agatcatacg ccgaacatgc tgcagcgggg tcaagaccac tgcgcatcgc ggtcactaca
      781 acccccatgg atgcaggtcg tgcagtggat ccggagtgcg tagccgcggt gaatcgcgta
      841 gccgagcgac tggcagagtt gggccatgtc gttgaggagg cggcccccga gcttgacgtg
      901 gcggcgttcg ataaagctta tcttgatgca tcttgctcaa gtctcgctga tgacgtactg
      961 ggtgcgagtg cgcagctggc cgtgcagcct agtcgtgaat atctggaggc cactactctg
     1021 gcatgtgtag aatacggtaa aaccctgagt gcctttgata tatttgccgc ggatcgtgtc
     1081 tttaaccaga caactcgcgc cgtagccggc tttttaaccc gttatgatgt tttactcacc
     1141 ccgacaacct cggcaccacc gatcccgtta gggcatctgg atgctgatga cgccagtttg
     1201 agtgcccgtg aatggtatga tcggatcttc gactatggct ccttcaccgc gctgttcaac
     1261 gtgacaggta tgcccgctat atcccttccg ctcgctgaga gcactgccgg tcttcctatc
     1321 ggtattcaat tcgccggtag atatggggac gaggcgacac tgctggcgct ggcaggtgat
     1381 cttgagcgcg ccatgccatg ggcagacaga cgccctgcgg tccacgttgg ccgactcgag
     1441 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1501 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1561 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1621 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1681 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1741 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1801 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1861 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1921 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1981 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2041 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2101 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2161 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2221 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2281 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2341 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2401 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2461 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2521 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2581 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2641 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2701 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2761 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2821 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2881 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2941 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3001 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3061 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3661 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3721 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3781 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3841 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3901 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3961 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4021 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4081 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4141 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4201 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4261 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4321 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4381 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4441 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4501 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4561 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4621 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4681 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4741 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4801 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4861 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4921 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4981 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     5041 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5101 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5221 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5281 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5341 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     5401 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5461 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5521 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5581 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5641 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5701 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5761 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5821 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5881 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5941 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6001 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6061 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6121 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6181 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6241 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6301 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6361 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6421 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     6481 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     6541 ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg
     6601 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
     6661 atatacatat g
//
