LOCUS       pMCSG35B.        7484 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding N-terminal 10xHis-MBP-TEV and C-terminal 
            TEV-6xHis plus TVMV protease. See also pMCSG35.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG35B
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7484)
  AUTHORS   Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan
            K, Kim Y, Joachimiak A.
  TITLE     Cleavable C-terminal His-tag vectors for structure determination.
  JOURNAL   J. Struct. Funct. Genomics 2010;11:31-9.
  PUBMED    20213425
REFERENCE   2  (bases 1 to 7484)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7484)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2010"; volume: "11"; pages: "31-9"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..7484
                     /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(208..225)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(232..252)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(263..283)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(305..1402)
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             complement(1430..1456)
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             complement(1457..1459)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1467..1489)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1504..1528)
                     /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(1529..1547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1778..1864)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(1900..2607)
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
     promoter        complement(2631..2704)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        2887..2964
                     /label=lacI promoter
     CDS             2965..4044
                     /label=lacI
                     /note="lac repressor"
     protein_bind    4060..4081
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4856..5044
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    5149..5291
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6239..7096)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7097..7200)
                     /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 tccgttaatg atgatgatgg tggtgcccgg cggattggaa
      241 gtacaggttc tccccgggag agactttgga agtacaggtt ctcggtacct gggatatcgt
      301 aatcagtctg cgcgtctttc agggcttcat cgacagtctg acgaccgctg gcggcgttga
      361 tcaccgcagt acgcacggca taccagaaag cggacatctg cgggatgttc ggcatgattt
      421 cacctttctg ggcgttttcc atggtggcgg caatacgtgg atctttcgcc aactcttcct
      481 cgtaagactt cagcgctacg gcacccagcg gtttgtcttt attaaccgct tccagacctt
      541 catcagtcag cagatagttt tcgaggaact cttttgccag ctctttgttc ggactggcgg
      601 cgttaatacc tgcgctcagc acgccaacga acggtttgga tggttgaccc ttgaaggtcg
      661 gcagtaccgt tacaccataa ttcactttgc tggtgtcgat gttggaccat gcccacgggc
      721 cgttgatggt catcgctgtt tcgcctttat taaaggcagc ttctgcgatg gagtaatcgg
      781 tgtctgcatt catgtgtttg tttttaatca ggtcaaccag gaaggtcaga cccgctttcg
      841 cgccagcgtt atccacgccc acgtctttaa tgtcgtactt gccgttttca tacttgaacg
      901 cataaccccc gtcagcagca atcagcggcc aggtgaagta cggttcttgc aggttgaaca
      961 tcagcgcgct cttacctttc gctttcagtt ctttatccag cgccgggatc tcttcccagg
     1021 tttttggcgg gttcggcagc agatctttgt tataaatcag cgataacgct tcaacagcga
     1081 tcgggtaagc aatcagcttg ccgttgtaac gtacggcatc ccaggtaaac ggatacagct
     1141 tgtcctggaa cgctttgtcc ggggtgattt cagccaacag gccagattga gcgtagccac
     1201 caaagcggtc gtgtgcccag aagataatgt cagggccatc gccagttgcc gcaacctgtg
     1261 ggaatttctc ttccagttta tccggatgct caacggtgac tttaattccg gtatctttct
     1321 cgaatttctt accgacttca gcgagaccgt tatagccttt atcgccgtta atccagatta
     1381 ccagtttacc ttcttcgatt ttggtaccca gatctacacc agaagagtgg tggtggtgat
     1441 gatgatgatg atggtgcata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     1501 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     1561 gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
     1621 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
     1681 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
     1741 gttgggcgcc atctccttgc atgctctagg gcggcggatt tgtcctactc aggagagcgt
     1801 tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     1861 tttgatgcct ctagcacgcg taccatggga tctttattag tccatgatgg cggcaacagt
     1921 ttttttggcc atgaagtcat cttccggcgc atcttcaacc agggtaaagg aaccccagct
     1981 gattttatcc gcgttgaatt tccagttttt gcaccaacca tccgcggcat ctagataagt
     2041 cgccacgaat ttttccggaa attccacgaa gtagttgcta ccgttggtgg tatgagtcag
     2101 gctgtggatg cccagaatgt tgccatcaat gatggaaact agtgggctgc cacactggcc
     2161 atctttagtg gtgatccagt gctgccagaa agaagtgtct tctttatgca caatgtgaga
     2221 ggattcagac accaggctcg agacgctttt ctgctgaaag ttggtggaca ccatgcacac
     2281 acgatctttg atggtcggct gacggaattt cagtttctgc gggaacggcg ggaagtcttt
     2341 agccattttg ataacgataa tgtcacggcc ttcaaccggt ttcatctgca gctgggtaga
     2401 gtttttgact ttgaattcac catgcatggt tttgatggtc agttcgccat tgttacgacg
     2461 aaacagatgc tggttggcaa tgatatacgg gccaaaacca atgccaaaca gacgttcact
     2521 atgaccatcc gaggagtttt ccagcaggca tacgcaagca gagatcggat taaaatcgcg
     2581 cacgcccttc agcaaagctt tagacatatg ttacctcctt aaatcaattc ggtcagtgcg
     2641 tcctgctgat gtgctcagta tctctatcac tgatagggat gtcaatctct atcactgata
     2701 gggactcgag gtgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2761 tgataataat ggtttcttag acgtcgcatg caccattcct tgcggcggcg gtgctcaacg
     2821 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     2881 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     2941 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     3001 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     3061 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     3121 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     3181 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     3241 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     3301 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     3361 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     3421 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     3481 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     3541 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     3601 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     3661 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     3721 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     3781 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     3841 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     3901 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     3961 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4021 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     4081 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     4141 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     4201 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     4261 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     4321 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     4381 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     4441 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     4501 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     4561 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     4621 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     4681 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     4741 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     4801 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     4861 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     4921 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     4981 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     5041 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     5101 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     5161 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     5221 gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     5281 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5881 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6121 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6181 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6541 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7081 aatgttgaat actcatactc ttcctttttc aaattattga agcatttatc agggttattg
     7141 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7201 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atgtgaacca tcaccctaat
     7261 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     7321 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     7381 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     7441 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
