LOCUS       pMCSG38.        6313 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector with a 6xHis-TEV leader and a 
            Tet-inducible TVMV protease cassette. See also pMCSG38C.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG38
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6313)
  AUTHORS   Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI.
  TITLE     A new vector for high-throughput, ligation-independent cloning 
            encoding a tobacco etch virus protease cleavage site.
  JOURNAL   Protein Expr. Purif. 2002;25:8-15.
  PUBMED    12071693
REFERENCE   2  (bases 1 to 6313)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6313)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2002"; volume: "25"; pages: "8-15"
COMMENT     SGRef: number: 2; 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..6313
                     /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(223..243)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(268..285)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(286..288)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(296..318)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(333..357)
                     /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(358..376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(607..693)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(729..1436)
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
     promoter        complement(1460..1533)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        1716..1793
                     /label=lacI promoter
     CDS             1794..2873
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2889..2910
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3685..3873
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3978..4120
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4306..4894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5068..5925)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5926..6029)
                     /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 acttccaata ttggattgga agtacaggtt
      241 ctcggtaccc agatctacac cagaagaatg atgatgatga tggtgcatat gtatatctcc
      301 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      361 atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc
      421 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
      481 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
      541 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgctctaggg
      601 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
      661 ccagtctttc gactgagcct ttcgttttat ttgatgcctc tagcacgcgt accatgggat
      721 ctttattagt ccatgatggc ggcaacagtt tttttggcca tgaagtcatc ttccggcgca
      781 tcttcaacca gggtaaagga accccagctg attttatccg cgttgaattt ccagtttttg
      841 caccaaccat ccgcggcatc tagataagtc gccacgaatt tttccggaaa ttccacgaag
      901 tagttgctac cgttggtggt atgagtcagg ctgtggatgc ccagaatgtt gccatcaatg
      961 atggaaacta gtgggctgcc acactggcca tctttagtgg tgatccagtg ctgccagaaa
     1021 gaagtgtctt ctttatgcac aatgtgagag gattcagaca ccaggctcga gacgcttttc
     1081 tgctgaaagt tggtggacac catgcacaca cgatctttga tggtcggctg acggaatttc
     1141 agtttctgcg ggaacggcgg gaagtcttta gccattttga taacgataat gtcacggcct
     1201 tcaaccggtt tcatctgcag ctgggtagag tttttgactt tgaattcacc atgcatggtt
     1261 ttgatggtca gttcgccatt gttacgacga aacagatgct ggttggcaat gatatacggg
     1321 ccaaaaccaa tgccaaacag acgttcacta tgaccatccg aggagttttc cagcaggcat
     1381 acgcaagcag agatcggatt aaaatcgcgc acgcccttca gcaaagcttt agacatatgt
     1441 tacctcctta aatcaattcg gtcagtgcgt cctgctgatg tgctcagtat ctctatcact
     1501 gatagggatg tcaatctcta tcactgatag ggactcgagg tgaagacgaa agggcctcgt
     1561 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcgcatgc
     1621 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     1681 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     1741 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     1801 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     1861 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     1921 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     1981 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     2041 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     2101 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     2161 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     2221 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     2281 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     2341 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     2401 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     2461 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     2521 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     2581 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     2641 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     2701 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     2761 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     2821 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     2881 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     2941 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     3001 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     3061 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     3121 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     3181 ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc
     3241 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     3301 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     3361 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     3421 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     3481 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     3541 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     3601 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     3661 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     3721 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     3781 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
     3841 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3901 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3961 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     4021 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatata tgcggtgtga
     4081 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     4141 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4201 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4261 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4321 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4381 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4441 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4501 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4561 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4621 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4681 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4741 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4801 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4861 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4921 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4981 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5041 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5101 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5161 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5221 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5281 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5341 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     5401 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5461 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5521 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5581 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5641 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5701 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5761 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5821 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5881 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5941 aattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6001 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     6061 taagcgttaa tgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     6121 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     6181 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     6241 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     6301 gtcccattcg cca
//
