LOCUS       40924_18601        7661 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pETMEOH500, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7661)
  AUTHORS   Woolston BM, King JR, Reiter M, Van Hove B, Stephanopoulos G.
  TITLE     Improving formaldehyde consumption drives methanol assimilation in 
            engineered E. coli
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7661)
  AUTHORS   Woolston BM, King JR, Reiter M, Van Hove B, Stephanopoulos G.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-APR-2018) Chemical Engineering, Massachusetts 
            Institute of Technology, 25 Ames Street, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 7661)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7661)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-APR-2018) Chemical Engineering, Massachusetts Institute of 
            Technology, 25 Ames Street, Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(667..688)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(704..1783)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(1784..1861)
                     /label=lacI promoter
     promoter        2170..2188
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2189..2213
                     /label=lac operator
                     /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             2228..2250
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2257..3429
                     /codon_start=1
                     /gene="mdh2"
                     /product="methanol dehydrogenase 2"
                     /label=mdh2
                     /note="from Cupriavidus necator"
                     /protein_id="AWJ76883.1"
                     /translation="MTHLNIANRVDSFFIPCVTLFGPGCVRETGVRARSLGARKALIVT
                     DAGLHKMGLSEVVAGHIREAGLQAVIFPGAEPNPTDVNVHDGVKLFEREECDFIVSLGG
                     GSSHDCAKGIGLVTAGGGHIRDYEGIDKSTVPMTPLISINTTAGTAAEMTRFCIITNSS
                     NHVKMVIVDWRCTPLIAIDDPSLMVAMPPALTAATGMDALTHAIEAYVSTAATPITDAC
                     AEKAIVLIAEWLPKAVANGDSMEARAAMCYAQYLAGMAFNNASLGYVHAMAHQLGGFYN
                     LPHGVCNAILLPHVSEFNLIAAPERYARIAELLGENIGGLSAHDAAKAAVSAIRTLSTS
                     IGIPAGLAGLGVKADDHEVMASNAQKDACMLTNPRKATLAQVMAIFAAAM"
     gene            2257..3429
                     /gene="mdh2"
                     /label=mdh2
     CDS             3460..4095
                     /codon_start=1
                     /gene="hps"
                     /product="hexulose phosphate synthase"
                     /label=hps
                     /note="from Bacillus methanolicus"
                     /protein_id="AWJ76884.1"
                     /translation="MELQLALDLVNIEEAKQVVAEVQEYVDIVEIGTPVIKIWGLQAVK
                     AVKDAFPHLQVLADMKTMDAAAYEVAKAAEHGADIVTILAAAEDVSIKGAVEEAKKLGK
                     KILVDMIAVKNLEERAKQVDEMGVDYICVHAGYDLQAVGKNPLDDLKRIKAVVKNAKTA
                     IAGGIKLETLPEVIKAEPDLVIVGGGIANQTDKKAAAEKINKLVKQGL"
     gene            3460..4095
                     /gene="hps"
                     /label=hps
     CDS             4116..4649
                     /codon_start=1
                     /gene="phi"
                     /product="phosphohexulose isomerase"
                     /label=phi
                     /note="from Methyloccus capsulatus"
                     /protein_id="AWJ76885.1"
                     /translation="MHQKLIIDKISGILAATDAGYDAKLTAMLDQASRIFVAGAGRSGL
                     VAKFFAMRLMHGGYDVFVVGEIVTPSIRKGDLLIVISGSGETETMLAFTKKAKEQGASI
                     ALISTRDSSSLGDLADSVFRIGSPELFGKVVGMPMGTVFELSTLLFLEATISHIIHEKG
                     IPEEEMRTRHANLE"
     gene            4116..4649
                     /gene="phi"
                     /label=phi
     CDS             4691..4708
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      4775..4822
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4859..5314
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5410..6222)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      6344..6932
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(7118..7260)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(7365..7553)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
       61 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
      121 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
      181 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
      241 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
      301 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
      361 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
      421 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
      481 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
      541 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
      601 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
      661 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
      721 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
      781 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
      841 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      901 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      961 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     1021 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     1081 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     1141 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     1201 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     1261 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     1321 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     1381 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     1441 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     1501 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     1561 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     1621 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     1681 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     1741 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1801 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1861 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1921 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1981 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     2041 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     2101 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     2161 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     2221 aaataatttt gtttaacttt aagaaggaga tataccatga cccacctgaa catcgctaat
     2281 cgcgtcgaca gcttcttcat tccctgcgtg accctcttcg gtccgggctg cgttcgcgaa
     2341 acgggcgtgc gcgccagatc actcggggcc aggaaggctc tcatcgtcac ggatgcaggc
     2401 ttgcacaaga tggggctctc cgaagtcgtc gcggggcaca ttcgcgaagc cgggctccag
     2461 gccgtcatct ttccgggtgc cgagcccaat cccaccgacg ttaacgttca cgacggcgtc
     2521 aagttgttcg agcgggaaga atgcgacttc atcgtttcgc tcggcggcgg ctcatcgcac
     2581 gactgcgcga aaggcatcgg cctcgttacc gccggaggcg gacatatccg cgactacgaa
     2641 ggcatcgaca aatcaacggt gccaatgacg ccgctgattt cgatcaacac gaccgctggc
     2701 actgctgcgg aaatgacacg cttttgcatc atcactaatt cgagcaatca tgtgaagatg
     2761 gtgatcgtcg actggcgttg cacgccatta atcgccatcg acgatccgag cctgatggtc
     2821 gcgatgccgc ccgccttgac ggcggcgacc ggcatggacg cgttgactca cgccatcgag
     2881 gcatacgttt ccaccgccgc cacgccaatt accgatgcct gtgcggagaa ggcgatcgtg
     2941 ctgatcgccg aatggctgcc caaagctgtc gcgaacgggg actcgatgga agcacgcgcg
     3001 gccatgtgct acgcccaata ccttgccggc atggccttca acaacgcatc actcggttac
     3061 gtgcacgcga tggcccatca actcggcggc ttctacaatt tgccccacgg cgtgtgcaac
     3121 gcgatcctgc tgccgcacgt gtcggaattc aacctcattg ccgcgccgga gcgctacgcg
     3181 agaatcgccg aactgctagg cgagaacatt gggggcttga gcgcgcatga cgccgccaaa
     3241 gctgccgtct cggcgatccg gaccctttcc acgtcgattg gcattccggc gggtctggcg
     3301 ggcctgggcg tcaaggcgga cgaccatgaa gtgatggcaa gcaatgcgca aaaggatgct
     3361 tgcatgctga cgaatccgcg caaggccacg ctggcgcaag tcatggcaat cttcgctgcg
     3421 gcgatgtaac gaagaaccaa gaaacgagga ggtcataaaa tggaattgca attagcctta
     3481 gatttagtaa acatcgaaga ggcaaagcag gtcgtagcgg aagtgcagga atacgtcgat
     3541 attgtcgaaa tcgggacacc tgtaatcaaa atctggggct tacaggcagt caaagccgta
     3601 aaggatgcct tcccacacct gcaagttttg gcagatatga agacgatgga tgccgccgct
     3661 tacgaggtcg ccaaggcagc tgagcatggt gctgacatcg taacaatctt agcagctgct
     3721 gaggacgttt caatcaaagg cgccgtagag gaagccaaga aactggggaa gaagattctt
     3781 gtggatatga tcgcagtcaa gaatctggag gaacgtgcca agcaggtgga tgaaatgggc
     3841 gttgattaca tttgcgtcca tgcgggatac gatttacagg cggtcggtaa gaacccactt
     3901 gacgatttaa aacgtattaa agcggtggtg aagaacgcga agacggctat cgccggaggc
     3961 attaagttag aaacgcttcc tgaagtaatc aaagctgagc cagatttagt aattgtcgga
     4021 ggaggtattg caaaccaaac cgataagaag gctgcagcag agaagattaa taaacttgta
     4081 aaacaaggtc tgtaaggatc caggaggaat taaccatgca tcagaaactc attatcgata
     4141 aaatctccgg tatcctggcc gcgactgatg ctggctacga cgctaaactc actgctatgc
     4201 tggatcaggc atcacgcatc tttgtggcgg gggcaggacg ttctgggctg gtggctaaat
     4261 tttttgccat gcgtctgatg catggcgggt atgacgtctt cgtagtcggc gagattgtta
     4321 ctcctagcat ccgtaaaggc gacctgctga ttgtaattag cggtagtggc gagactgaga
     4381 ccatgcttgc gtttaccaaa aaagctaagg agcagggggc ctcaatcgcc ctgattagta
     4441 ctcgtgattc gtccagcctg ggggatttgg cagactcagt cttccgtatc ggttcgcccg
     4501 agctctttgg taaggttgtt ggcatgccta tgggtacggt attcgaactg agcacacttc
     4561 tgtttttaga agcaaccatc agtcatatca ttcatgaaaa aggtatcccg gaagaagaga
     4621 tgcgcacccg tcacgctaat ttggaatgac gaattcgagc tccgtcgaca agcttgcggc
     4681 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     4741 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4801 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     4861 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4921 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4981 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5041 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     5101 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5161 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5221 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5281 gcgaatttta acaaaatatt aacgtttaca atttcaggtg gcacttttcg gggaaatgtg
     5341 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     5401 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     5461 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     5521 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     5581 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     5641 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     5701 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     5761 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     5821 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     5881 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     5941 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     6001 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     6061 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     6121 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     6181 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     6241 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     6301 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6361 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6421 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6481 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6541 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6601 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6661 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6721 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6781 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6841 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6901 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6961 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7021 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7081 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     7141 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     7201 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     7261 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     7321 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     7381 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     7441 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     7501 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     7561 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     7621 ctcacgatac gggttactga tgatgaacat gcccggttac t
//
