LOCUS       Exported                6508 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Modification of CYP2C9 and 2C19 s for bacterial expression .
ACCESSION   .
VERSION     .
KEYWORDS    CYP2C9 in vector pCW ori+
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  TITLE     Goldstein CYP2C9 and CYP2C9 in pOri+ for bacterial expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6508)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6508
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        129..159
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    167..183
                     /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)."
     promoter        238..266
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    274..290
                     /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)."
     promoter        333..361
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    369..385
                     /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)."
     primer_bind     complement(2065..2083)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      2145..2600
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2232..2251)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2442..2463
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2633..2737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2738..3598
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2956..2975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3769..4357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4258..4277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4511..4528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4543..4684
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4610..4629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4770..4792
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4805..5266)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVLPRAFR"
     primer_bind     5215..5237
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5229..5246
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5230..5246
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5262..5278)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5262..5278)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5286..5302
                     /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)."
     promoter        complement(5310..5340)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(5389..6471)
                     /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     6433..6452
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6472..41)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
ORIGIN
        1 catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtataatgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag gatcgatcca tcgatgagct tactccccat ccccctgttg
      241 acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag
      301 gaaacaggat cagcttactc cccatccccc tgttgacaat taatcatcgg ctcgtataat
      361 gtgtggaatt gtgagcggat aacaatttca cacaggaaac aggatccatc gatgcttagg
      421 aggtcatatg gctctgttat tagcagtttt tctctgtctc tcatgtttgc ttctcctttc
      481 actctggaga cagagctctg ggagaggaaa actccctcct ggccccactc ctctcccagt
      541 gattggaaat atcctacaga taggtattaa ggacatcagc aaatccttaa ccaatctctc
      601 aaaggtctat ggccctgtgt tcactctgta ttttggcctg aaacccatag tggtgctgca
      661 tggatatgaa gcagtgaagg aagccctgat tgatcttgga gaggagtttt ctggaagagg
      721 cattttccca ctggctgaaa gagctaacag aggatttgga attgttttca gcaatggaaa
      781 gaaatggaag gagatccggc gtttctccct catgacgctg cggaattttg ggatggggaa
      841 gaggagcatt gaggaccgtg ttcaagagga agcccgctgc cttgtggagg agttgagaaa
      901 aaccaaggcc tcaccctgtg atcccacttt catcctgggc tgtgctccct gcaatgtgat
      961 ctgctccatt attttccata aacgttttga ttataaagat cagcaatttc ttaacttaat
     1021 ggaaaagttg aatgaaaaca tcaagatttt gagcagcccc tggatccaga tctgcaataa
     1081 tttttctcct atcattgatt acttcccggg aactcacaac aaattactta aaaacgttgc
     1141 ttttatgaaa agttatattt tggaaaaagt aaaagaacac caagaatcaa tggacatgaa
     1201 caaccctcag gactttattg attgcttcct gatgaaaatg gagaaggaaa agcacaacca
     1261 accatctgaa tttactattg aaagcttgga aaacactgca gttgacttgt ttggagctgg
     1321 gacagagacg acaagcacaa ccctgagata tgctctcctt ctcctgctga agcacccaga
     1381 ggtcacagct aaagtccagg aagagattga acgtgtgatt ggcagaaacc ggagcccctg
     1441 catgcaagac aggagccaca tgccctacac agatgctgtg gtgcacgagg tccagagata
     1501 cattgacctt ctccccacca gcctgcccca tgcagtgacc tgtgacatta aattcagaaa
     1561 ctatctcatt cccaagggca caaccatatt aatttccctg acttctgtgc tacatgacaa
     1621 caaagaattt cccaacccag agatgtttga ccctcatcac tttctggatg aaggtggcaa
     1681 ttttaagaaa agtaaatact tcatgccttt ctcagcagga aaacggattt gtgtgggaga
     1741 agccctggcc ggcatggagc tgtttttatt cctgacctcc attttacaga actttaacct
     1801 gaaatctctg gttgacccaa agaaccttga caccactcca gttgtcaatg gatttgcctc
     1861 tgtgccgccc ttctaccagc tgtgcttcat tcctgtctga agaagagcag atggcctggc
     1921 tgctgctgtg cagtccctgc agctctcttt cctctggggc attatccatc tttaagctta
     1981 tcgatgataa gctgtcaaac atgagcagat ctgagcccgc ctaatgagcg ggcttttttt
     2041 tcagatctgc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2101 tgataataat ggtttcttag acgatgcgtc aaagcaacca tagtacgcgc cctgtagcgg
     2161 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2221 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2281 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagagctt tacggcacct
     2341 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2401 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2461 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     2521 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2581 aatattaacg tttacaattt catcgtcagg tggcactttt cggggaaatg tgcgcggaac
     2641 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2701 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2761 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2821 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2881 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2941 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     3001 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3061 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3121 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3181 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3241 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     3301 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3361 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3421 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3481 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3541 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3601 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3661 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3721 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3781 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3841 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3901 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3961 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4021 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4081 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4141 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4201 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4261 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4321 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4381 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4441 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4501 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4561 ccttacgcat ctgtgcggta tttcacaccg cataatggtg cactctcagt acaatctgct
     4621 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4681 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4741 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4801 gtcatcaccg aaacgcgcga ggcagaacgc catcaaaaat aattcgcgtc tggccttcct
     4861 gtagccagct ttcatcaaca ttaaatgtga gcgagtaaca acccgtcgga ttctccgtgg
     4921 gaacaaacgg cggattgacc gtaatgggat aggtcacgtt ggtgtagatg ggcgcatcgt
     4981 aaccgtgcat ctgccagttt gaggggacga cgacagtatc ggcctcagga agatcgcact
     5041 ccagccagct ttccggcacc gcttctggtg ccggaaacca ggcaaagcgc cattcgccat
     5101 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     5161 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     5221 cacgacgttg taaaacgacg gccagtgaat ccgtaatcat ggtcatagct gtttcctgtg
     5281 tgaaattgtt atccgctcac aattccacac attatacgag ccggaagcat aaagtgtaaa
     5341 gcctggggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5401 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5461 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5521 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5581 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5641 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5701 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5761 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5821 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5881 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     5941 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6001 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6061 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6121 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6181 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6241 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6301 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6361 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6421 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6481 cctgaattga ctctcttccg ggcgctat
//
