LOCUS       sequence-426074-        6026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for ThcCut1 gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6026)
  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 6026)
  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: ThcCut1_pET29b
FEATURES             Location/Qualifiers
     source          1..6026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             796..813
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(866..884)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      880..927
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      964..1419
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1051..1070)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1261..1282
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1511..2326)
                     /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(1511..2326)
                     /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     2234..2253
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2318..2337
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2448..3036
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2937..2956
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3190..3207
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3222..3361
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3287..3306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3447..3469
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3463..3654)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4463..5545)
                     /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     5507..5526
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5546..5623)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5829..5848
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5936..5955
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5936..5954
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5955..5979
                     /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             5994..6016
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gcgaatcctt acgaacgcgg accgaacccg actgatgcgt tactggaggc aagcagcggt
       61 ccgttttccg tgtctgagga aaatgtatct cgtctttcag cctccggctt tgggggcggt
      121 acaatctatt atccgcgtga aaataacact tacggcgccg ttgccattag cccgggctat
      181 acgggcacgg aagcgtccat tgcctggctt ggcgagcgta tagccagcca cggatttgtg
      241 gtgattacca ttgatacaat caccacactg gaccagccag acagccgtgc ggagcagtta
      301 aatgctgctc tcaatcatat gataaataga gcgagctcta ctgttcgcag ccgaatcgat
      361 agttcacgtt tagctgttat gggtcattct atgggcggtg gtggtaccct gcgcttggcg
      421 tcacaaagac cagatttaaa ggctgcaatc ccgttgacgc cgtggcattt aaacaaaaat
      481 tggtcgagtg tcacggtgcc gacacttatt atcggggctg atcttgatac cattgcacct
      541 gtggccaccc acgccaagcc gttttacaat tcgttaccgt cctcgatctc taaagcctat
      601 cttgagctcg atggcgcgac gcactttgcc cctaatatac caaacaaaat cataggcaaa
      661 tattctgtag cttggttaaa gcgctttgtt gataacgata cacgttatac acaatttctg
      721 tgcccggggc cccgcgacgg cctgttcggg gaagttgagg aatatcgttc tacgtgccca
      781 ttcgcgttgc tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      841 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      901 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      961 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1021 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1081 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1141 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1201 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1261 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1321 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1381 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1441 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1501 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1561 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1621 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1681 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1741 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1801 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1861 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     1921 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1981 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2041 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2101 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2161 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2221 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2281 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2341 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2401 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2461 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2521 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2581 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2641 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2701 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2761 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2821 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2881 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2941 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3001 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3061 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3121 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3181 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3241 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3301 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3361 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3421 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3481 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3541 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3601 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3661 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3721 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3781 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     3841 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     3901 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3961 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4021 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4081 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4141 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4201 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4261 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4321 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4381 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4441 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4501 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4561 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4621 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4681 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4741 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4801 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     4861 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4921 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4981 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5041 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5101 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5161 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5221 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5281 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5341 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5401 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5461 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5521 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5581 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5641 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5701 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5761 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5821 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     5881 ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata
     5941 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     6001 aactttaaga aggagatata catatg
//
