LOCUS       40924_18551        6400 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pETHISmdh, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6400)
  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  JOURNAL   Unpublished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 6400)
  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 6400)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6400)
  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            "Unpublished"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..6400
                     /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             2608..2625
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      3862..3909
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3946..4401
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(4497..5309)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      5431..6019
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(6205..6347)
                     /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 taccatgcat catcatcatc atcacaccca cctgaacatc
     2641 gctaatcgcg tcgacagctt cttcattccc tgcgtgaccc tcttcggtcc gggctgcgtt
     2701 cgcgaaacgg gcgtgcgcgc cagatcactc ggggccagga aggctctcat cgtcacggat
     2761 gcaggcttgc acaagatggg gctctccgaa gtcgtcgcgg ggcacattcg cgaagccggg
     2821 ctccaggccg tcatctttcc gggtgccgag cccaatccca ccgacgttaa cgttcacgac
     2881 ggcgtcaagt tgttcgagcg ggaagaatgc gacttcatcg tttcgctcgg cggcggctca
     2941 tcgcacgact gcgcgaaagg catcggcctc gttaccgccg gaggcggaca tatccgcgac
     3001 tacgaaggca tcgacaaatc aacggtgcca atgacgccgc tgatttcgat caacacgacc
     3061 gctggcactg ctgcggaaat gacacgcttt tgcatcatca ctaattcgag caatcatgtg
     3121 aagatggtga tcgtcgactg gcgttgcacg ccattaatcg ccatcgacga tccgagcctg
     3181 atggtcgcga tgccgcccgc cttgacggcg gcgaccggca tggacgcgtt gactcacgcc
     3241 atcgaggcat acgtttccac cgccgccacg ccaattaccg atgcctgtgc ggagaaggcg
     3301 atcgtgctga tcgccgaatg gctgcccaaa gctgtcgcga acggggactc gatggaagca
     3361 cgcgcggcca tgtgctacgc ccaatacctt gccggcatgg ccttcaacaa cgcatcactc
     3421 ggttacgtgc acgcgatggc ccatcaactc ggcggcttct acaatttgcc ccacggcgtg
     3481 tgcaacgcga tcctgctgcc gcacgtgtcg gaattcaacc tcattgccgc gccggagcgc
     3541 tacgcgagaa tcgccgaact gctaggcgag aacattgggg gcttgagcgc gcatgacgcc
     3601 gccaaagctg ccgtctcggc gatccggacc ctttccacgt cgattggcat tccggcgggt
     3661 ctggcgggcc tgggcgtcaa ggcggacgac catgaagtga tggcaagcaa tgcgcaaaag
     3721 gatgcttgca tgctgacgaa tccgcgcaag gccacgctgg cgcaagtcat ggcaatcttc
     3781 gctgcggcga tgtaatgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     3841 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     3901 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     3961 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4021 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4081 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4141 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4201 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4261 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4321 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4381 aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4441 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     4501 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     4561 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     4621 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     4681 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     4741 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     4801 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     4861 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     4921 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     4981 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     5041 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     5101 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     5161 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     5221 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     5281 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     5341 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5401 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5461 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5521 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     5581 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5641 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5701 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5761 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5821 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5881 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5941 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6001 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6061 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6121 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     6181 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     6241 tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6301 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     6361 caacacccgc tgacgcgccc tgacgggctt gtctgctccc
//
