LOCUS       sequence-453971-        6022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses codon-optimized PETase from Ideonella sakaiensis in E. 
            coli without native signal sequence and with a C-terminal his-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6022)
  AUTHORS   Carter LM, MacFarlane CE, Karlock SP, Sen T, Kaar JL, Berberich JA, 
            Boock JT
  TITLE     Increased cytoplasmic expression of PETase enzymes in E. coli.
  JOURNAL   Microb Cell Fact. 2024 Nov 25;23(1):319. doi: 
            10.1186/s12934-024-02585-w.
  PUBMED    39582006
REFERENCE   2  (bases 1 to 6022)
  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: "Microb Cell
            Fact."; date: "2024-11-25"; volume: "23(1):319. doi"; pages: " 
            10.1186/s12934-024-02585-w"
COMMENT     Sequence Label: pET24a isPETase
FEATURES             Location/Qualifiers
     source          1..6022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..74
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(68..259)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1068..2150)
                     /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     2112..2131
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2151..2228)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2434..2453
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2537..2556
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2537..2555
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2556..2580
                     /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             2596..2616
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3423..3440
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3493..3511)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3507..3554
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3591..4046
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3678..3697)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3888..3909
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4138..4953)
                     /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(4138..4953)
                     /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     4861..4880
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4945..4964
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5075..5663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5564..5583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5817..5834
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5849..5988
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5914..5933)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
       61 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
      121 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
      181 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
      241 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
      301 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
      361 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
      421 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
      481 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
      541 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
      601 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
      661 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
      721 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
      781 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
      841 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
      901 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
      961 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     1021 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     1081 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1141 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     1201 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     1261 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     1321 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     1381 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     1441 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     1501 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     1561 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     1621 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     1681 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     1741 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     1801 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     1861 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     1921 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     1981 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     2041 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     2101 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     2161 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     2221 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     2281 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     2341 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     2401 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     2461 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     2521 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     2581 cctctagaaa taattttgtt taactttaag aaggaggagt acatatgcag actaatccat
     2641 acgcccgcgg acctaaccct acggcggcga gtttggaggc ttctgctggt ccgttcacag
     2701 ttcgttcttt tacggtctcg cgtccaagcg ggtacggcgc tggaaccgtc tactatccca
     2761 ctaacgctgg aggcacagta ggagctatcg ccattgtccc cggctacact gctcgccaaa
     2821 gcagtatcaa atggtggggt ccgcgcttgg cctcacatgg gtttgttgtg attaccatcg
     2881 acactaattc cacattggac cagccatcct cacgttcttc acaacaaatg gctgccttac
     2941 gccaagtggc gtcgcttaat gggacttcta gttcaccaat ctacggtaag gtagatactg
     3001 ctcgcatggg agttatggga tggagcatgg gcggtggggg gagccttatt tccgcagcga
     3061 ataacccatc cctgaaggct gccgcacctc aggccccatg ggattcgtca actaatttca
     3121 gctcggtcac cgtccctact cttattttcg cttgtgaaaa cgacagtatc gcacctgtta
     3181 attcatcggc actgccaatc tatgattcca tgtcccgtaa cgctaagcaa ttcctggaga
     3241 ttaacggggg aagtcactcg tgcgctaata gtgggaacag taatcaagct cttatcggga
     3301 agaagggtgt tgcatggatg aaacgcttta tggacaacga cacacgttat agtacctttg
     3361 catgcgaaaa tcctaattct acgcgcgtga gtgacttccg cacggcaaat tgcagcctcg
     3421 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     3481 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3541 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     3601 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3661 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3721 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3781 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3841 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3901 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3961 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4021 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     4081 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     4141 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     4201 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     4261 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     4321 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     4381 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     4441 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4501 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4561 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     4621 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     4681 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     4741 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     4801 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     4861 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     4921 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     4981 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5041 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5101 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5161 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5221 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5281 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5341 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5401 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5461 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5521 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5581 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5641 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5701 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5761 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5821 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5881 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5941 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     6001 ccaacacccg ctgacgcgcc ct
//
