pETMEOH500 vector (V006452)

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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  JOURNAL   Unpublished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            "Unpublished"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"