LOCUS       sequence-425822-        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for LCC-ICCG gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  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 6140)
  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: LCC-ICCG_pET21b
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             8..25
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(78..96)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      92..139
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      176..631
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(263..282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     473..494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        657..761
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(762..830,831..1622)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(980..999)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1793..2381
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2282..2301
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2535..2552
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2567..2706
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2632..2651)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2792..2814
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2808..2999)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3808..4890)
                     /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     4852..4871
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4891..4968)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5174..5193
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5277..5296
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5277..5295
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5296..5320
                     /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             5335..5357
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
       61 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      121 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
      181 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      241 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      301 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      361 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
      421 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      481 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
      541 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      601 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
      661 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
      721 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
      781 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
      841 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
      901 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
      961 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1021 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1081 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1141 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1201 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1261 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     1321 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1381 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     1441 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     1501 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     1561 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     1621 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     1681 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     1741 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     1801 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     1861 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     1921 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1981 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2041 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2101 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2161 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2221 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2281 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2341 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2401 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     2461 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     2521 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     2581 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     2641 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     2701 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     2761 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2821 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     2881 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     2941 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3001 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3061 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3121 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3181 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3241 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3301 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3361 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     3421 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     3481 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     3541 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     3601 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     3661 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     3721 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     3781 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3841 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3901 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     3961 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4021 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4081 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4141 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4201 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4261 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4321 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4381 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     4441 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4501 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     4561 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     4621 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     4681 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     4741 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     4801 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     4861 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     4921 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     4981 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5041 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5101 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     5161 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     5221 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     5281 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     5341 taactttaag aaggagatat acatatgtct aacccgtacc agcgcggacc gaacccgacc
     5401 cgttctgcgt taaccgctga tggtccgttt tccgtggcta cctacaccgt ttctcgtctg
     5461 tccgtttccg gttttggtgg tggtgttatc tactatccga ctggtacctc tctgaccttc
     5521 ggcggtatcg cgatgtcccc gggttacacc gctgatgctt cctctctggc gtggctgggt
     5581 cgtcgcctgg cgagccacgg ttttgttgtt ctggttatca acacgaactc tcgtttcgac
     5641 ggcccggact cccgtgcctc gcaactgtct gctgcgctga actacctgcg tacgtcgtca
     5701 ccttcagcgg tccgtgcacg cctggatgcc aatcgtctgg ctgtggcggg tcacagcatg
     5761 ggcggtggcg gtaccctgcg tattgctgaa cagaacccgt ccctgaaagc tgcagtgcca
     5821 ctgactccgt ggcataccga caaaacgttc aacaccagtg ttccggtact gatcgtaggc
     5881 gcagaagcgg acaccgtagc accggtttcc cagcacgcaa tcccgttcta ccagaacctg
     5941 ccgagcacca ctccaaaagt atacgttgaa ctgtgcaacg cctcgcacat tgctccgaac
     6001 tcgaacaacg ctgcgattag cgtgtacacc atctcctgga tgaaactgtg ggttgataac
     6061 gatacccgtt atcgccaatt cctgtgtaac gtgaacgatc cggctctctg cgattttcgt
     6121 accaacaacc gtcattgcca
//
