LOCUS       Exported                7661 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pETMEOH500, complete sequence.
ACCESSION   MH173315
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  JOURNAL   Unpublished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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7661
                     /organism="Expression vector pETMEOH500"
                     /mol_type="other DNA"
                     /db_xref="taxon:2161722"
     CDS             complement(701..1792)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="AWJ76882.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     gene            complement(701..1792)
                     /gene="lacI"
                     /label=lacI
     CDS             complement(701..1783)
                     /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"
     promoter        complement(1784..1861)
                     /gene="lacI"
                     /label=lacI promoter
     regulatory      2170..2189
                     /regulatory_class="promoter"
                     /note="T7"
     promoter        2170..2188
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2189..2213
                     /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)."
     misc_binding    2189..2211
                     /label=lac repressor binding site
                     /bound_moiety="lac repressor"
                     /note="lacO"
     RBS             2228..2250
                     /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
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4775..4822
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      4776..4822
                     /regulatory_class="terminator"
                     /note="T7"
     rep_origin      4859..5314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(5407..6222)
                     /codon_start=1
                     /gene="kanR"
                     /product="kanamycin resistance protein"
                     /label=kanR
                     /protein_id="AWJ76886.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(5407..6222)
                     /gene="kanR"
                     /label=kanR
     rep_origin      6344..6932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    7118..7260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(7362..7553)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"