LOCUS       sequence-321466-        7313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-21b(+) based plasmid for expression of the putative MHET 
            hydrolase from Comamonas thiooxydans (Genbank WP_080747404.1) with 
            C-terminal His tag, codon optimized for expression in E. coli K12..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7313)
  AUTHORS   Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, Pardo 
            I, Topuzlu E, Anderson JJ, Austin HP, Dominick G, Johnson CW, Rorrer
            NA, Szostkiewicz CJ, Copie V, Payne CM, Woodcock HL, Donohoe BS, 
            Beckham GT, McGeehan JE
  TITLE     Characterization and engineering of a two-enzyme system for plastics
            depolymerization.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Oct 13;117(41):25476-25485. doi: 
            10.1073/pnas.2006753117. Epub 2020 Sep 28.
  PUBMED    32989159
REFERENCE   2  (bases 1 to 7313)
  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"; doi: 
            "10.1073/pnas.2006753117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-10-13- 13"; volume: "117"; issue: "41"; pages: 
            "25476-25485"
COMMENT     Sequence Label: pCJ199
FEATURES             Location/Qualifiers
     source          1..7313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        493..597
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(598..666,667..1458)
                     /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(816..835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1629..2217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2118..2137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2371..2388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2403..2542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2468..2487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2628..2650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2644..2835)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3644..4726)
                     /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     4688..4707
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4727..4804)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5010..5029
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5113..5132
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5113..5131
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5132..5156
                     /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             5171..5193
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7157..7174
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7227..7245)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7241..7288
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      601 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      661 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      721 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      781 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      841 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      901 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      961 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1021 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1081 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1141 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1201 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1261 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1321 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1381 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1441 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1501 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1561 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1621 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1681 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1741 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     1801 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1861 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1921 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1981 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2041 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2101 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2161 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2221 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2281 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2341 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2401 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     2461 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2521 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2581 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2641 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     2701 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     2761 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     2821 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     2881 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     2941 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3001 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3061 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3121 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3181 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3241 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     3301 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     3361 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     3421 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     3481 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     3541 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     3601 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     3661 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3721 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     3781 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     3841 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     3901 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     3961 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     4021 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     4081 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     4141 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     4201 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     4261 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     4321 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     4381 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     4441 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     4501 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     4561 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     4621 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     4681 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     4741 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     4801 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     4861 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     4921 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     4981 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     5041 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     5101 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     5161 tagaaataat tttgtttaac tttaagaagg agatatacat atgttcgtac gcaacgccga
     5221 ccgtgccaag aattgtatgc gcgcaccttt acgcgtattc ccactcaagg atacttttag
     5281 cgcccagtgt gcgaatgttt cggtctggat taccagcagc gtaccaccgc tccgcgagcg
     5341 tcacatggat cgccgcgtga cgcgccgcga tttaatgcaa actcgcatct tattaatgct
     5401 cattgcagcc actggcgtgg cagcgtgtgg cggagacggt ggttccacac ctgccgcgca
     5461 aaatccccct ttgcccctgg ccagtcgtgc ggcttgcgaa gcttttcaag gcaatagcaa
     5521 tagtatcgcg tggccccatc gcgcaaccgt tgtggaagtg gccacttggc acgaagcaga
     5581 gcctgcgaat gccacagcag cggcgacgcc cgagcactgt gagatttccg gcgccattgc
     5641 tcgccgcacc ggaattgatg gatatcctta cgagattaag tttcgcttac gtatgccctc
     5701 agaatggaat ggtcgcttct ttatggaagg ggggggtgga accaatgggt cattgagtgc
     5761 cgctacaggg tcccttggcg gtggacaaac tgcgtcggcc ttgagtcgta attttgcaac
     5821 tattgcaacc gatggtggtc atgataatgc tgtcaacaat aatcctgatg cgctcggcac
     5881 tgtcgctttt ggcatggacc ctcaagcgcg cattgatatg ggatataatt cctacgacca
     5941 ggtgacccaa gcgggaaagg cggccgtagc gcaattttat ggccgtgccc cggataaaag
     6001 ctattttatt ggctgtagcg aaggtgggcg cgaggggatg atgttgtccc aacgctttcc
     6061 gagtcattat gacggaattg tggcgggagc accgggctac caactcccaa aggcgggcat
     6121 ttctggcgca tggacgacgc aaagtctggc accagcagcg gtgggcgtgg atgctcaaaa
     6181 tgtacctttg atcaataagg cgttttcgga tgtcgattta catcttcttt cacgcggcat
     6241 tcttggtact tgcgatgcct tggatggact cagcgatgga attgtgaacg acttccgtgc
     6301 ctgtcaagcc gcctttgacc ctgccactgc gttgaatccc gacaccagtc aacccttaca
     6361 atgcactggt gctaagacgc ctgattgctt aagtgccgcc caagtcactg gcattaaacg
     6421 cgccatgggt gggcctgtgg acagcgccgg tgcggcatta tataatcgct gggcatggga
     6481 ccctggcatg tcggggctca atggcacctc ttacaatcag ggatggcgct cttggtggtt
     6541 agggagctac gcatccagca ctaataatgc ccaacgcgtc aatggctttt ccgcccgctc
     6601 ttggttggtt gattttgcga cgccgcctga accaatgcca gtcacacagg ttgctgctcg
     6661 catgatgaac ttcaactttg acaccgaccc gcctaagatt cgcgcgacta gtggcccctt
     6721 tactccatcg tctatggagt ggcatggtgc aacgagcact aatcttgcgg ccttccgcga
     6781 ccgcggtggg aagctgatgc tctatcatgg catgtcagat gccgcgttct ccgcattaga
     6841 caccgcggat tactacgagc gtttaggtgc cgcgatgccg ggcgccgccg gcttcgcacg
     6901 tcttttctta gttccaggta tgaatcattg tagtggtgga cccggcactg atcgttttga
     6961 tatgcttact ccccttgtgg cctgggtgga acgtgataag gcgccagatc aagttagcgc
     7021 ttgggcaggc acaccgggct atttcggcgc aaccgcccgt acacgccccc tttgtccata
     7081 cccccaaatc gcacgttata agggctctgg tgatatcaat gccgaggcat cgtttgtgtg
     7141 tgtggcccca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     7201 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     7261 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gat
//
