LOCUS       sequence-426033-        6288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for Tt-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 6288)
  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 6288)
  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: Tt-NylC_pET28a
FEATURES             Location/Qualifiers
     source          1..6288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1003..1020
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1073..1091)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1087..1134
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1171..1626
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1258..1277)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1468..1489
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1718..2533)
                     /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(1718..2533)
                     /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     2441..2460
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2525..2544
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2655..3243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3144..3163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3397..3414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3429..3568
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3494..3513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3654..3676
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3670..3861)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4670..5752)
                     /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     5714..5733
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5753..5830)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6036..6055
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6139..6158
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6139..6157
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6158..6182
                     /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             6197..6219
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6238..6255
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6265..6282
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 acccccagtg ccgaaccaag actcgccgct ccctctgcgt tcgcacctga cgtggctggc
       61 aatgatgatt tgccgctcac tccccaggcg tcgcctggtc gtggcgtcgt acccttcgat
      121 cttcccggtg tctatgtggg cacggcagag tatgctgaag gtcccacggg ctgcaccgtc
      181 gtccacgtac cggctggggc tcggaccgct gtggacgccc gtggtggcgc ggtgggcctc
      241 tcggggggat acgactttaa ccatgcaatt tgtttagcgg gcggatctgt atacgggctg
      301 gcggcggcag ccggagtaag cgatgaattg ttagcgcgtc ggcggaatca ggtccggtgg
      361 gatgaccttc agattgtttc gggggctgtc atctatgatt tttcagcgag agataatgcc
      421 gtatcgcccg acgccgcttt aggtcgcgcg gccctgcgaa acgccaaaga aggggagttt
      481 cccgtaggtc gctgcggcgc aggaagatct gctacagcag gcaaagtgga atttggtaga
      541 gccgaattca cgggtcaagg ggccgctttc cgcgcctatg gcgacgtcaa agtcttggtg
      601 gcaaccgtcg ttaacagcgt tggtgtcgtt gttgaccgcg acggagcgat agtacgtggt
      661 aattatcatt ctgctgaagg cgttcggcgg caccccgctg aagattacgc cgccatgatc
      721 ggccgtcccg atgcgccggc gccggtccct gggaatacga ccttaacggt actggtgacc
      781 aatgtgcgcc tgcccgagcg cgaacttaac caactgggac gccaggtaca tgggtcaatg
      841 caacgcggga tacagccttt ccatacagag aatgatggag atgtgttatt tacaatgacc
      901 actgacgaag tggatttgac cggggttcgt agcactggcc tggctactct ggcggccgag
      961 gtagcatggg atgcgatatt ggacgccgcg cgttgactcg agcaccacca ccaccaccac
     1021 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1081 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1141 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1201 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1261 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1321 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1381 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1441 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1501 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1561 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1621 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1681 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1741 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1801 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1861 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1921 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1981 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2041 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2101 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2161 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2221 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2281 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2341 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2401 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2461 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2521 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2581 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2641 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2701 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2761 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2821 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2881 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2941 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3001 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3061 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3121 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3181 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3241 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3301 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3361 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3421 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3481 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3541 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3601 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3661 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3721 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3781 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     3841 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     3901 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     3961 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4021 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4081 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4141 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4201 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4261 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4321 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4381 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4441 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4501 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4561 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4621 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4681 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4741 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4801 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4861 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4921 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4981 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5041 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5101 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5161 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5221 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5281 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5341 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5401 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5461 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5521 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5581 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5641 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5701 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5761 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5821 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5881 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5941 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6001 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6061 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     6121 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     6181 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
     6241 catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatg
//
