LOCUS       pMCSG48.        6741 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with an 8xHis-NusA-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG48.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6741)
  AUTHORS   Donnelly MI, Zhou M, Millard CS, Clancy S, Stols L, Eschenfeldt WH, 
            Collart FR, Joachimiak A.
  TITLE     An expression vector tailored for large-scale, high-throughput 
            purification of recombinant proteins.
  JOURNAL   Protein Expr. Purif. 2006;47:446-54.
  PUBMED    16497515
REFERENCE   2  (bases 1 to 6741)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6741)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2006"; volume: "47"; pages: "446-54"
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..6741
                     /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(253..1710)
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
     CDS             complement(1717..1740)
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             complement(1741..1743)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1751..1773)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1788..1812)
                     /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(1813..1831)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2144..2221
                     /label=lacI promoter
     CDS             2222..3301
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3317..3338
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4113..4301
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    4406..4548
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4734..5322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5496..6353)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6354..6457)
                     /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 ctcgttgttg ttattacggg cagccataat cagtgctccg gctttttcgt cggtcaaccc
      301 ttcgatatca gccagatcat caatgccctg ttcggcgaga tcttccagcg tacaaacgcc
      361 acgggcggcc agtttgaatg ccaaatcacg atctacccct tcaaggttca gcagatcgtc
      421 agccggtttg ttatcaccga ggctttcttc ctgggcctgt gcaatggtgg ccagtgcatt
      481 tttagcacgc tcgcgcagtg cttcaacggt cggctcatca aggccttcga tttccaacag
      541 ctctttcatc ggcacatagg ccaattcttc cagcgtcgag aagccttctt ctaccagaac
      601 agtcgcgaag tcttcgtcga tgtcgagata tttggtgaag gtgtcgatcg ctgcgtgcgc
      661 ttccgcctga tgcttagctt gcaggtcgtc aacggtcatc acgttgagtt cccaaccgct
      721 cagttgcgaa gccagacgca cgttctgacc gttacggcca atcgcctgcg ccagattacc
      781 ggcttcaacg gcgatgtcca tggtgtgttt atcttcatcc accacgatag aagcaacgtc
      841 tgccggtgcc attgcgttaa tcacgaactg cgccgggtta tcatcccaca ggacgatatc
      901 gatacgctcg ccacccagtt cagtagacac cgcctgaaca cgcgcgccac gcatacctac
      961 gcaagcacct accggatcga tacgtttatc gttggttttc accgcgattt tcgcacgaga
     1021 acccggatcg cgagccgctg ctttaatttc aatcacttct tcgccgattt ctggcacttc
     1081 aatacggaac agttcgatca gcatttccgg cttggaacga gtgacgaaca gttgcgcgcc
     1141 acgcgcttcc gggcgaacgg aatagagcac gccacgaacg cggtcgccag ggcggaagtt
     1201 ttcacgcggc agcatatctt cgcgcaggat cacggcttca gcgttgttgc ccagatccag
     1261 agagatgttg tcgcggttta cttttttcac cacgccggtg atgatttcac cttcgtgttc
     1321 acggaactga tcaaccacca tcgcacgttc ggcttcacgc actttctgca cgataacctg
     1381 ttttgccgtc tgggtagtga tacggtcaaa ggtaacagac tcaatctgat cttcaacgta
     1441 atcgcccagg ttcaggcttt catcttcata acgtgcggct tcaagggtga tttccttggt
     1501 cggctgggtg acttcatcaa caactaacca gcgacggaaa gtgtcaaaat caccgctttt
     1561 gcgatcgatc tgtacgcgga cgtcgatctc ttgttcatat tttttctttg ttgctgtcgc
     1621 cagcgcgctt tccaatgctt cgaaaatctt ctcgcgaggt agcgcctttt cattggatac
     1681 ggcttcaact acagccaaaa tttctttgtt agaagaatga tgatgatgat gatgatggtg
     1741 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1801 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
     1861 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     1921 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     1981 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     2041 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     2101 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     2161 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     2221 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     2281 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     2341 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     2401 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     2461 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     2521 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     2581 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     2641 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     2701 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     2761 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     2821 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     2881 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2941 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     3001 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     3061 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     3121 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     3181 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     3241 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3301 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     3361 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     3421 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     3481 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     3541 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     3601 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     3661 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     3721 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     3781 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     3841 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     3901 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     3961 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     4021 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     4081 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     4141 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     4201 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     4261 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4321 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4381 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     4441 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg
     4501 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4561 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4621 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4681 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4741 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4801 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4861 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4921 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4981 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5041 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5101 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5161 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5221 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5281 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5341 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5401 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5461 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5521 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5581 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5641 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5701 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5761 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     5821 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5881 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5941 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6001 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6061 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6121 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6181 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6241 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6301 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6361 ctttttcaaa ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6421 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6481 ctaaattgta agcgttaatg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6541 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6601 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6661 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6721 cagggcgcgt cccattcgcc a
//
