LOCUS       pMCSG31.        5822 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector with an MBP-TVMV-6xHis-TEV leader and a 
            Tet-inducible TVMV protease cassette.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG31
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5822)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5822)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..5822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..91
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     RBS             130..152
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             184..891
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
     terminator      927..1013
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        1244..1262
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1263..1287
                     /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             1318..1323
                     /note="ribosome binding site"
     CDS             1332..2432
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     protein_bind    2442..2466
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2469..2489
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
     CDS             2490..2507
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             2532..2552
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             2618..2635
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      2702..2749
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2818..2841
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     rep_origin      2932..3390
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3416..3519
                     /label=AmpR promoter
     CDS             3520..4377
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             4387..5010
                     /label=TetR
                     /note="tetracycline repressor TetR"
     rep_origin      5166..5754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt
       61 tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct
      121 agaaataatt ttgtttaact ttaagaagga gatatacaaa tgggagaccg cggtcccgaa
      181 ttgatgtcta aagctttgct gaagggcgtg cgcgatttta atccgatctc tgcttgcgta
      241 tgcctgctgg aaaactcctc ggatggtcat agtgaacgtc tgtttggcat tggttttggc
      301 ccgtatatca ttgccaacca gcatctgttt cgtcgtaaca atggcgaact gaccatcaaa
      361 accatgcatg gtgaattcaa agtcaaaaac tctacccagc tgcagatgaa accggttgaa
      421 ggccgtgaca ttatcgttat caaaatggct aaagacttcc cgccgttccc gcagaaactg
      481 aaattccgtc agccgaccat caaagatcgt gtgtgcatgg tgtccaccaa ctttcagcag
      541 aaaagcgtct cgagcctggt gtctgaatcc tctcacattg tgcataaaga agacacttct
      601 ttctggcagc actggatcac cactaaagat ggccagtgtg gcagcccact agtttccatc
      661 attgatggca acattctggg catccacagc ctgactcata ccaccaacgg tagcaactac
      721 ttcgtggaat ttccggaaaa attcgtggcg acttatctag atgccgcgga tggttggtgc
      781 aaaaactgga aattcaacgc ggataaaatc agctggggtt cctttaccct ggttgaagat
      841 gcgccggaag atgacttcat ggccaaaaaa actgttgccg ccatcatgga ctaataaaga
      901 tcccatggta cgcgtgctag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
      961 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc
     1021 cctagagcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     1081 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     1141 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     1201 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata
     1261 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1321 gagatataca tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag
     1381 gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca
     1441 ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg
     1501 gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt
     1561 tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg
     1621 ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga
     1681 tttataacaa agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg
     1741 ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact
     1801 tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt
     1861 acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg
     1921 ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg
     1981 cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg
     2041 acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca
     2101 aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg
     2161 caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag
     2221 acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac
     2281 gtattgccgc caccatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga
     2341 tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga
     2401 ctgtcgatga agccctgaaa gacgcgcaga ctaattcgat cacaagtttg tacaaaaaag
     2461 caggctcgga aaccgtgcgt ttccagtctc accaccatca tcatcactct tctggcgtgg
     2521 atctgggtac cgagaacctg tacttccaat ccaatattgg aagtggataa cggatccgaa
     2581 ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga
     2641 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2701 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2761 aactataccc ggggatccct cgaggtcgac ctgcaggggg accatggtct cagcgcttgg
     2821 agccacccgc agttcgaaaa ataataagct tgacctgtga agtgaaaaat ggcgcacatt
     2881 gtgcgacatt ttttttgtct gccgtttacc gctactgcgt cacggatctc cacgcgccct
     2941 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3001 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3061 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3121 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3181 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3241 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3301 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3361 ttaacaaaat cacattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3421 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3481 accctgataa atgcttcaat aatttgaaaa aggaagagta tgagtattca acatttccgt
     3541 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3601 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3661 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3721 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3781 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3841 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3901 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3961 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4021 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     4081 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attgatagac
     4141 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4201 tttattgctg ataaatctgg agccggtgag cgtggctctc gcggtatcat tgcagcactg
     4261 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4321 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtag
     4381 gaattaatga tgtctcgttt agataaaagt aaagtgatta acagcgcatt agagctgctt
     4441 aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag
     4501 cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt
     4561 gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat
     4621 tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca
     4681 aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta
     4741 gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgcagtg
     4801 gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa
     4861 agggaaacac ctactactga tagtatgccg ccattattac gacaagctat cgaattattt
     4921 gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta
     4981 gaaaaacaac ttaaatgtga aagtgggtct taaaagcagc ataacctttt tccgtgatgg
     5041 taacttcact agtttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5101 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5161 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5221 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5281 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5341 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5401 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5461 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5521 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5581 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5641 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5701 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5761 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgacccga
     5821 ca
//
