LOCUS       sequence-349670-        6254 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes codon optimized putative thermophilic PET hydrolase enzyme 
            for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6254)
  AUTHORS   Erickson E, Gado JE, Avilan L, Bratti F, Brizendine RK, Cox PA, Gill
            R, Graham R, Kim DJ, Konig G, Michener WE, Poudel S, Ramirez KJ, 
            Shakespeare TJ, Zahn M, Boyd ES, Payne CM, DuBois JL, Pickford AR, 
            Beckham GT, McGeehan JE
  TITLE     Sourcing thermotolerant poly(ethylene terephthalate) hydrolase 
            scaffolds from natural diversity.
  JOURNAL   Nat Commun. 2022 Dec 21;13(1):7850. doi: 10.1038/s41467-022-35237-x.
  PUBMED    36543766
REFERENCE   2  (bases 1 to 6254)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2022-12-21"; volume: "13(1):7850. doi"; pages: " 
            10.1038/s41467-022-35237-x"
COMMENT     Sequence Label: pEE006
FEATURES             Location/Qualifiers
     source          1..6254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             905..922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(975..993)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      989..1036
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1073..1528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1160..1179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1370..1391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1554..1658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1659..1727,1728..2519)
                     /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(1877..1896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2690..3278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3179..3198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3432..3449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3464..3603
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3529..3548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3689..3711
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3705..3896)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4705..5787)
                     /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     5749..5768
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5788..5865)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6071..6090
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6174..6193
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6174..6192
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6193..6217
                     /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             6232..6254
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg cagtctccgg cacaatctag cgcaccgact gtggaactcg attctggtgc
       61 catcgcgggc tctaccgctg atggcgttgt gtcctttaaa ggcatcccat acgctgctcc
      121 gccggtgggt aatctgcgtt ggcgcgctcc acagccggtt gcgagctgga ctggtgttcg
      181 tgctgcgact gaatatggct acgactgcat ccagctgccg ctggaaggtg atgcggctgc
      241 atctggtggt gaaatgtctg aagattgtct ggtcctgaac gtatggcgcc cggctgaaat
      301 tgcaccgggc gaacgcctgc cagttctggt ttggatccac gggggtggct ttctgaatgg
      361 tagcgcagcg gccccgatct atgacggcac cgcgtttgca cagcagggtc tggtagtagt
      421 aagcttcaac taccgtctgg gtcgtctggg tttctttgcg cacccggctc tgaccgcggc
      481 gaacgagggt ccgctgggta actacgggct gatggaccag atcgctgcgc tggaatgggt
      541 gcagcgtaat atcgctgcat tcggtggtga cccagcacgt attaccctca tgggccaatc
      601 ggcgggcggt attagcgtaa tgtaccacct gaccgcacct gaatctcaag gtctgttcca
      661 ccaggcggct gttctgtccg gtggcggtcg tacttatctg ctgggtctgc gtaacctgcg
      721 tgaaagcacc gatgcgctgc caagcgcaga acagtctggt ctggctttcg gtcgtcgttt
      781 cggcattcgt ggccgcggcc gtgctgctct gcgttctctg agatctctga gcgccgaaga
      841 agttaacggt gatctgtcga tggcagctct ggttgagaaa ccggccgatt atgcaggtct
      901 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
      961 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1021 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1081 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1141 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1201 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1261 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1321 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1381 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1441 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1501 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     1561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1621 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1681 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1741 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1801 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1861 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1921 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1981 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2041 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2101 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2161 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     2221 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2281 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2341 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2401 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2461 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2521 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2581 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2641 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2701 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2761 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2821 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2881 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2941 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3001 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3061 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3121 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3181 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3241 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3301 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3361 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3421 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3481 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3541 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3601 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3661 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3721 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3781 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3841 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3901 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3961 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4021 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4081 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4141 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4201 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4261 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4321 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4381 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4441 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4501 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4561 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4621 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4681 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4741 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4801 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4861 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4921 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4981 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5041 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5101 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5161 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5221 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5281 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5341 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5401 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5461 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5521 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5581 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5641 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5701 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5761 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5821 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5881 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5941 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     6001 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6061 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6121 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     6181 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     6241 ctttaagaag gaga
//
