LOCUS       40924_18596        6382 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pETmdh, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6382)
  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  JOURNAL   Unpublished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 6382)
  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 6382)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6382)
  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            "Unpublished"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..6382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(52..240)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(1015..1036)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(1052..2131)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(2132..2209)
                     /label=lacI promoter
     promoter        2518..2536
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2537..2561
                     /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             2576..2598
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2605..3777
                     /codon_start=1
                     /gene="mdh2"
                     /product="methanol dehydrogenase 2"
                     /label=mdh2
                     /note="from Cupriavidus necator"
                     /protein_id="AWJ76877.1"
                     /translation="MTHLNIANRVDSFFIPCVTLFGPGCVRETGVRARSLGARKALIVT
                     DAGLHKMGLSEVVAGHIREAGLQAVIFPGAEPNPTDVNVHDGVKLFEREECDFIVSLGG
                     GSSHDCAKGIGLVTAGGGHIRDYEGIDKSTVPMTPLISINTTAGTAAEMTRFCIITNSS
                     NHVKMVIVDWRCTPLIAIDDPSLMVAMPPALTAATGMDALTHAIEAYVSTAATPITDAC
                     AEKAIVLIAEWLPKAVANGDSMEARAAMCYAQYLAGMAFNNASLGYVHAMAHQLGGFYN
                     LPHGVCNAILLPHVSEFNLIAAPERYARIAELLGENIGGLSAHDAAKAAVSAIRTLSTS
                     IGIPAGLAGLGVKADDHEVMASNAQKDACMLTNPRKATLAQVMAIFAAAM"
     gene            2605..3777
                     /gene="mdh2"
                     /label=mdh2
     terminator      3844..3891
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3928..4383
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(4479..5291)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      5413..6001
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(6187..6329)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
       61 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
      121 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
      181 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
      241 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
      301 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      361 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
      421 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
      481 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
      541 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
      601 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
      661 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
      721 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
      781 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
      841 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
      901 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
      961 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     1021 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1081 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1141 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1201 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1261 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     1321 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     1381 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     1441 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     1501 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     1561 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     1621 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     1681 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     1741 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     1801 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     1861 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     1921 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     1981 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     2041 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     2101 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2161 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     2221 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     2281 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     2341 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     2401 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     2461 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     2521 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     2581 ttaactttaa gaaggagata taccatgacc cacctgaaca tcgctaatcg cgtcgacagc
     2641 ttcttcattc cctgcgtgac cctcttcggt ccgggctgcg ttcgcgaaac gggcgtgcgc
     2701 gccagatcac tcggggccag gaaggctctc atcgtcacgg atgcaggctt gcacaagatg
     2761 gggctctccg aagtcgtcgc ggggcacatt cgcgaagccg ggctccaggc cgtcatcttt
     2821 ccgggtgccg agcccaatcc caccgacgtt aacgttcacg acggcgtcaa gttgttcgag
     2881 cgggaagaat gcgacttcat cgtttcgctc ggcggcggct catcgcacga ctgcgcgaaa
     2941 ggcatcggcc tcgttaccgc cggaggcgga catatccgcg actacgaagg catcgacaaa
     3001 tcaacggtgc caatgacgcc gctgatttcg atcaacacga ccgctggcac tgctgcggaa
     3061 atgacacgct tttgcatcat cactaattcg agcaatcatg tgaagatggt gatcgtcgac
     3121 tggcgttgca cgccattaat cgccatcgac gatccgagcc tgatggtcgc gatgccgccc
     3181 gccttgacgg cggcgaccgg catggacgcg ttgactcacg ccatcgaggc atacgtttcc
     3241 accgccgcca cgccaattac cgatgcctgt gcggagaagg cgatcgtgct gatcgccgaa
     3301 tggctgccca aagctgtcgc gaacggggac tcgatggaag cacgcgcggc catgtgctac
     3361 gcccaatacc ttgccggcat ggccttcaac aacgcatcac tcggttacgt gcacgcgatg
     3421 gcccatcaac tcggcggctt ctacaatttg ccccacggcg tgtgcaacgc gatcctgctg
     3481 ccgcacgtgt cggaattcaa cctcattgcc gcgccggagc gctacgcgag aatcgccgaa
     3541 ctgctaggcg agaacattgg gggcttgagc gcgcatgacg ccgccaaagc tgccgtctcg
     3601 gcgatccgga ccctttccac gtcgattggc attccggcgg gtctggcggg cctgggcgtc
     3661 aaggcggacg accatgaagt gatggcaagc aatgcgcaaa aggatgcttg catgctgacg
     3721 aatccgcgca aggccacgct ggcgcaagtc atggcaatct tcgctgcggc gatgtaatga
     3781 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     3841 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     3901 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     3961 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4021 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4081 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4141 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4201 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4261 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4321 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4381 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4441 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     4501 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     4561 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     4621 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     4681 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     4741 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     4801 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     4861 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     4921 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     4981 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     5041 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     5101 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     5161 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     5221 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     5281 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     5341 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5521 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6121 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6181 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata
     6241 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     6301 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     6361 cctgacgggc ttgtctgctc cc
//
