LOCUS       Exported                6400 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pETHISmdh, complete sequence.
ACCESSION   MH173314
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  JOURNAL   Unpublished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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6400
                     /organism="Expression vector pETHISmdh"
                     /mol_type="other DNA"
                     /db_xref="taxon:2161721"
     CDS             complement(49..240)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1049..2140)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="AWJ76879.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     gene            complement(1049..2140)
                     /gene="lacI"
                     /label=lacI
     CDS             complement(1049..2131)
                     /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(2132..2209)
                     /gene="lacI"
                     /label=lacI promoter
     regulatory      2518..2537
                     /regulatory_class="promoter"
                     /note="T7"
     promoter        2518..2536
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2537..2561
                     /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    2537..2559
                     /label=lac repressor binding site
                     /bound_moiety="lac repressor"
                     /note="lacO"
     RBS             2576..2598
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2605..3795
                     /codon_start=1
                     /gene="mdh2"
                     /product="methanol dehydrogenase 2"
                     /label=mdh2
                     /note="from Cuprivaidus necator with N-terminal HIS tag"
                     /protein_id="AWJ76880.1"
                     /translation="MHHHHHHTHLNIANRVDSFFIPCVTLFGPGCVRETGVRARSLGAR
                     KALIVTDAGLHKMGLSEVVAGHIREAGLQAVIFPGAEPNPTDVNVHDGVKLFEREECDF
                     IVSLGGGSSHDCAKGIGLVTAGGGHIRDYEGIDKSTVPMTPLISINTTAGTAAEMTRFC
                     IITNSSNHVKMVIVDWRCTPLIAIDDPSLMVAMPPALTAATGMDALTHAIEAYVSTAAT
                     PITDACAEKAIVLIAEWLPKAVANGDSMEARAAMCYAQYLAGMAFNNASLGYVHAMAHQ
                     LGGFYNLPHGVCNAILLPHVSEFNLIAAPERYARIAELLGENIGGLSAHDAAKAAVSAI
                     RTLSTSIGIPAGLAGLGVKADDHEVMASNAQKDACMLTNPRKATLAQVMAIFAAAM"
     gene            2605..3795
                     /gene="mdh2"
                     /label=mdh2
     CDS             2608..2625
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3862..3909
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      3863..3909
                     /regulatory_class="terminator"
                     /note="T7"
     rep_origin      3946..4401
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(4494..5309)
                     /codon_start=1
                     /gene="kanR"
                     /product="kanamycin resistance protein"
                     /label=kanR
                     /protein_id="AWJ76881.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(4494..5309)
                     /gene="kanR"
                     /label=kanR
     rep_origin      5431..6019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    6205..6347
                     /label=bom
                     /note="basis of mobility region from pBR322"