LOCUS       sequence-425804-        6155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for HotPETase gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6155)
  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 6155)
  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: HotPETase_pET21b
FEATURES             Location/Qualifiers
     source          1..6155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(77..95)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      91..138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      175..630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(262..281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     472..493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        656..760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(761..829,830..1621)
                     /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(979..998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1792..2380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2281..2300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2534..2551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2566..2705
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2631..2650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2791..2813
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2807..2998)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3807..4889)
                     /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     4851..4870
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4890..4967)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5173..5192
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5276..5295
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5276..5294
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5295..5319
                     /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             5334..5356
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
       61 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      121 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      181 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      241 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      301 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      361 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      421 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      481 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      541 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      601 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      661 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
      721 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
      781 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
      841 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
      901 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
      961 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     1021 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1081 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1141 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1201 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     1261 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     1321 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     1381 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     1441 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     1501 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     1561 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     1621 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     1681 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     1741 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1801 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1861 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1921 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1981 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2041 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2101 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2161 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2221 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2281 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2341 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2401 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2461 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2521 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2581 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     2641 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     2701 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2761 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2821 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     2881 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     2941 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3001 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3061 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3121 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3181 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3241 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3301 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3361 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3421 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     3481 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3541 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3601 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3661 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     3721 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     3781 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3841 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3901 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3961 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4021 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4081 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4141 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4201 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4261 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4321 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4381 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4441 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4501 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4561 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4621 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4681 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4741 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4801 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4861 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4921 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     4981 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5041 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5101 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5161 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5221 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     5281 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     5341 aactttaaga aggagatata catatgcaga ctaatccgta tgcacgaggc ccgaacccga
     5401 ccgcagccag cttagaggcg tcggccgggc cgttcacagt acgttcattt actgtcgctc
     5461 gcccagtcgg ttacggtgcg ggtacagtgt attatccaac caatgcaggt gggactgtag
     5521 gggctatcgc catcgtgccg gggtacactg cgacccagtc cagtattaat tggtggggtc
     5581 cccggttggc gtctcatggc tttgtcgtga ttaccataga tacaaactcc accctcgata
     5641 aaccagaatc tagatcttcg caacagatgg ccgctttacg ccaggttgcg tcactgaatg
     5701 gtacatcgtc ttcacctatc tatggaaaag tggatacggc tcgcggaggt gttatggggt
     5761 ggtctatggg cggaggagga agtttgattt ctgccgctaa caatccaagc ttaaaagcag
     5821 ccgcggtgat ggcgccttgg cattccagta ctaatttcag cagcgtaaca gtacccacgt
     5881 taatttttgc ctgtgagaac gaccggatag cccctgtaaa agaatatgcc ttaccgatct
     5941 acgactctat gagtttaaat gcaaagcagt ttctggaaat ctgtggtggc agtcattcct
     6001 gcgcctgttc cggcaacagc aaccaggcat tgatcggcat gaaaggcgtc gcatggatga
     6061 aacgtttcat ggataacgac acccggtaca gtcagtttgc ttgcgaaaac ccaaattcaa
     6121 ccgcagtgtg cgactttcgt accgcgaatt gttcc
//
