LOCUS       pMCSG30.        8314 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector with a 6xHis-TEV-MBP leader and a SacB 
            negative selection marker, for high-throughput purification of 
            recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG30
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8314)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8314)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SfiI to 
            remove the SacB cassette, and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..8314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(258..1676)
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        complement(1677..2122)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(2147..3244)
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             complement(3251..3271)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(3296..3313)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(3314..3316)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(3324..3346)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(3361..3385)
                     /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)."
     promoter        complement(3386..3404)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3717..3794
                     /label=lacI promoter
     CDS             3795..4874
                     /label=lacI
                     /note="lac repressor"
     protein_bind    4890..4911
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             5686..5874
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    5979..6121
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6307..6895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7069..7926)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7927..8030)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgttatcc acttccaatg ttatttttca ccgacgacaa
      241 gggccgccgt ggccttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg
      301 acaacagatg ttttcttgcc tttgatgttc agcaggaagc ttggcgcaaa cgttgattgt
      361 ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc
      421 gcttgaggta cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt
      481 tttaacacaa ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca
      541 taaccaagca tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg
      601 gagtcagtga acaggtacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca
      661 tctgttactg tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg
      721 tttagctcaa tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt
      781 tgcagaagtt tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg
      841 ttaaataaag attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact
      901 aagtatttgt ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct
      961 gagctgtagt tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg
     1021 tcaaagattg atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat
     1081 acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg
     1141 tagaataaac ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt
     1201 tggtctttta ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca
     1261 gcgtttttcc agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat
     1321 gtgtcatccg catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag
     1381 tttgcgacag tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt
     1441 gcagaagaga tatttttaat tgtggacgaa tcgaattcag gaacttgata tttttcattt
     1501 ttttgctgtt cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt
     1561 tccttatatg gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc
     1621 agtgcggtag taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt
     1681 catgtctcct tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa
     1741 gatggcaagt tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa
     1801 agtatacact ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg
     1861 cgcgatttac ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt
     1921 ttcctctttt gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact
     1981 aaaaaatcta tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca
     2041 taaagttgcc tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa
     2101 tagtgaacgg caggtatatg tgttaggccg ccgtggccgc tgcgttggtt tgagcagcgg
     2161 caagagcagc atcgacagtc tgacgaccgc tggcggcgtt gatcaccgca gtacgcacgg
     2221 cataccagaa agcggacatc tgcgggatgt tcggcatgat ttcacctttc tgggcgtttt
     2281 ccatggtggc ggcaatacgt ggatctttcg ccaactcttc ctcgtaagac ttcagcgcta
     2341 cggcacccag cggtttgtct ttattaaccg cttccagacc ttcatcagtc agcagatagt
     2401 tttcgaggaa ctctttcgcc agctctttgt tcggactggc ggcgttaata cctgcgctca
     2461 gcacgccaac gaacggtttg gatggttgac ccttgaaggt cggcagtacc gttacaccat
     2521 aattcacttt gctggtgtcg atgttggacc atgcccacgg gccgttgatg gtcatcgctg
     2581 tttcgccttt attaaaggca gcttctgcga tggagtaatc ggtgtctgca ttcatgtgtt
     2641 tgtttttaat caggtcaacc aggaaggtca gacccgcttt cgcgccagcg ttatccacgc
     2701 ccacgtcttt aatgtcgtac ttgccgtttt catacttgaa cgcataaccc ccgtcagcag
     2761 caatcagcgg ccaggtgaag tacggttctt gcaggttgaa catcagcgcg ctcttacctt
     2821 tcgctttcag ttctttatcc agcgccggga tctcttccca ggtttttggc gggttcggca
     2881 gcagatcttt gttataaatc agcgataacg cttcaacagc gatcgggtaa gcaatcagct
     2941 tgccgttgta acgtacggca tcccaggtaa acggatacag cttgtcctgg aacgctttgt
     3001 ccggggtgat ttcagccaac aggccagatt gagcgtagcc accaaagcgg tcgtgtgccc
     3061 agaagataat gtcagggcca tcgccagttg ccgcaacctg tgggaatttc tcttccagtt
     3121 tatccggatg ctcaacggtg actttaattc cggtatcttt ctcgaatttc ttaccgactt
     3181 cagcgagacc gttatagcct ttatcgccgt taatccagat taccagttta ccttcttcga
     3241 ttttggcatt ggattggaag tacaggttct cggtacccag atctacacca gaagaatgat
     3301 gatgatgatg gtgcatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
     3361 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     3421 atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     3481 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     3541 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     3601 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     3661 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     3721 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     3781 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     3841 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     3901 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     3961 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4021 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4081 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4141 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4201 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4261 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4321 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4381 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     4441 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     4501 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     4561 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     4621 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     4681 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     4741 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     4801 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     4861 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     4921 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     4981 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     5041 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     5101 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     5161 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     5221 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     5281 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     5341 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     5401 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     5461 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     5521 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     5581 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     5641 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     5701 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     5761 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     5821 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     5881 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     5941 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     6001 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     6061 tgcaccatat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     6121 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6181 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6241 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6301 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6361 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6421 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6481 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6541 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6601 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6661 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6721 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     6781 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6841 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6901 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6961 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7021 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7081 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7141 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7201 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7261 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7321 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7381 tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7441 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7501 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7561 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7621 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7681 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7741 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7801 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7861 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7921 actcatactc ttcctttttc aaattattga agcatttatc agggttattg tctcatgagc
     7981 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8041 cgaaaagtgc cacctaaatt gtaagcgtta atgtgaacca tcaccctaat caagtttttt
     8101 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     8161 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     8221 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     8281 taatgcgccg ctacagggcg cgtcccattc gcca
//
