LOCUS       Exported                6233 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pET-21b(+) based plasmid for expression of PETase from Ideonella 
            sakaiensis 201-F6 (Genbank GAP38373.1) with W159H and S238F 
            mutations, codon optimized for expression in E. coli K12.
ACCESSION   .
VERSION     .
KEYWORDS    pET21b(+)-Is-PETase-W159H-S238F
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6233)
  AUTHORS   Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, 
            Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, 
            Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, 
            Woodcock HL, McGeehan JE, Beckham GT
  TITLE     Characterization and engineering of a plastic-degrading aromatic 
            polyesterase.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 May 8;115(19):E4350-E4357. doi: 
            10.1073/pnas.1718804115. Epub 2018 Apr 17.
  PUBMED    29666242
REFERENCE   2  (bases 1 to 6233)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6233
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(43..61)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      57..104
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      141..596
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(228..247)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     438..459
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        622..726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             727..1587
                     /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(945..964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1758..2346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2247..2266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2500..2517
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2532..2671
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2597..2616)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2757..2779
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2773..2964)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3773..4855)
                     /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     4817..4836
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4856..4933)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5139..5158
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5242..5261
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5242..5260
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5261..5285
                     /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             5300..5322
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6206..6223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
       61 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
      121 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
      181 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
      241 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      301 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
      361 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
      421 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
      481 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
      541 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
      601 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
      661 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      721 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      781 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      841 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      901 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      961 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     1021 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     1081 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     1141 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     1201 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     1261 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     1321 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     1381 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     1441 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     1501 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1561 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1621 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1681 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1741 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1801 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1861 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     1921 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1981 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2041 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2101 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2161 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2221 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2281 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2341 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2401 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2461 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2521 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2581 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     2641 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     2701 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     2761 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     2821 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     2881 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     2941 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3001 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3061 tgcccggtta ctggaacgtt gtgagggtaa acaacttgcg gtatggatgc ggcgggacca
     3121 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     3181 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     3241 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     3301 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     3361 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagta gcacgatcat
     3421 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     3481 ggcgggacca gtgacgaagg cttgagcgat ggcgtgcaag attccgaata ccgcaagcga
     3541 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     3601 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     3661 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     3721 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     3781 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3841 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     3901 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     3961 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4021 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4081 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4141 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4201 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4261 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4321 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4381 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4441 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4501 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4561 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     4621 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     4681 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     4741 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     4801 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     4861 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     4921 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     4981 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5041 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5101 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     5161 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     5221 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     5281 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgaacttccc
     5341 ccgtgcctcg cgccttatgc aggctgctgt gctgggcggc cttatggccg tttccgcagc
     5401 ggccaccgcg cagaccaatc cgtatgcgcg cggccccaac cctaccgccg cctcgttgga
     5461 agccagcgcg ggacccttta ccgttcgtag ctttaccgtt agccgtccgt ccggatatgg
     5521 tgcagggacc gtctattacc caaccaatgc aggcggcacc gttggcgcga ttgcaatcgt
     5581 ccccgggtac accgcgcgtc aaagcagcat taagtggtgg ggtccgcgct tagctagcca
     5641 tggctttgtg gttattacca tcgatacgaa cagcactcta gaccagccca gcagccgtag
     5701 ctcgcaacag atggccgcgc ttcgtcaagt tgcgagcttg aacgggacca gcagtagccc
     5761 gatttacgga aaggtcgata ctgcccgcat gggtgtgatg ggccactcaa tggggggcgg
     5821 cggttcactt attagcgccg cgaacaaccc gagtttaaaa gcagcggcac cgcaggcgcc
     5881 atgggactct tcaaccaact tcagcagtgt taccgtgccg acgctgattt tcgcgtgcga
     5941 gaatgatagc attgcaccgg tgaacagcag cgcgctgccg atttatgata gcatgtcccg
     6001 caacgcaaaa cagtttctgg aaattaacgg cggtagccac ttctgtgcca actctgggaa
     6061 cagcaaccag gcactgatcg gaaaaaaagg ggttgcatgg atgaaacgat tcatggataa
     6121 tgacacccgt tactcaacct tcgcctgtga gaatcccaac agcacacgcg tgtcggattt
     6181 tcgcaccgcg aactgttccc tcgagcacca ccatcaccac cactgagatc cgg
//
