LOCUS       sequence-426026-        6029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for ThcCut2 gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6029)
  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 6029)
  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: ThcCut2 _pET29b
FEATURES             Location/Qualifiers
     source          1..6029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             799..816
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(869..887)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      883..930
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      967..1422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1054..1073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1264..1285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1514..2329)
                     /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(1514..2329)
                     /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     2237..2256
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2321..2340
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2451..3039
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2940..2959
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3193..3210
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3225..3364
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3290..3309)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3450..3472
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3466..3657)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4466..5548)
                     /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     5510..5529
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5549..5626)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5832..5851
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5939..5958
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5939..5957
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5958..5982
                     /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             5997..6019
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gccaatccct acgaacgggg ccctaatcct accgatgccc tgttagaagc ccgtagcggg
       61 ccatttagtg tttccgaaga gcgtgccagc cgctttggcg cggatggatt tgggggaggg
      121 acgatttatt atccgcgtga gaataataca tatggggctg ttgctatttc acctggctac
      181 actgggaccc aggcaagtgt tgcctggctg ggtgagcgga ttgcgagtca tggctttgtt
      241 gttatcacga ttgatactaa cacgactttg gaccaacctg attcgagagc gcgccaactt
      301 aatgccgctt tagactatat gattaacgat gccagctcgg ccgttcgttc aagaattgat
      361 tccagtcgac ttgcagttat gggccactca atgggtgggg gtggtacatt gcgtctggcg
      421 tcccaacggc cggatttaaa ggcggcgata ccattaacac cttggcattt gaacaagaac
      481 tggagtagcg ttcgggttcc gacgttgatc ataggcgctg acttagatac aattgcaccg
      541 gtgttaacgc atgctcgccc gttttataat tctctcccta ctagtatatc caaagcgtat
      601 ttagagctgg atggcgctac gcattttgcc ccgaatattc cgaataaaat tattgggaaa
      661 tactctgttg cgtggctgaa gcgttttgta gacaatgata cacgatacac acagtttctg
      721 tgccctggac cccgtgatgg gctttttgga gaagttgagg aatatcggtc tacatgtcca
      781 tttgcgctgg agctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
      841 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      901 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      961 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1021 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1081 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1141 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1201 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1261 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1321 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1381 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1441 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1501 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     1561 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     1621 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     1681 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     1741 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     1801 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     1861 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     1921 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     1981 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     2041 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     2101 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2161 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     2221 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     2281 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     2341 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     2401 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2461 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2521 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2581 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2641 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2701 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2761 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2821 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2881 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2941 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3001 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3061 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3121 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3181 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3241 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3301 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3361 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3421 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3481 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3541 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3601 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3661 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3721 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     3781 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     3841 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     3901 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     3961 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4021 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4081 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4141 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4201 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4261 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4321 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4381 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4441 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4501 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4561 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4621 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4681 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4741 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4801 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4861 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4921 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4981 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5041 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5101 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5161 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5221 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5281 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5341 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5401 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5461 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5521 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5581 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5641 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5701 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5761 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5821 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     5881 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
     5941 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     6001 tttaacttta agaaggagat atacatatg
//
