LOCUS       pMCSG46.        6759 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a 6xHN-NusA-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG46
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  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 6759)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6759)
  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..6759
                     /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(259..1716)
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
     CDS             complement(1717..1752)
                     /label=6xHN
                     /note="6x(His-Asn) affinity tag"
     CDS             complement(1759..1761)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1769..1791)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1806..1830)
                     /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(1831..1849)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2162..2239
                     /label=lacI promoter
     CDS             2240..3319
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3335..3356
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4131..4319
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    4424..4566
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4752..5340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5514..6371)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6372..6475)
                     /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 ctcgttctcg ttgttgttat tacgggcagc cataatcagt gctccggctt tttcgtcggt
      301 caacccttcg atatcagcca gatcatcaat gccctgttcg gcgagatctt ccagcgtaca
      361 aacgccacgg gcggccagtt tgaatgccaa atcacgatct accccttcaa ggttcagcag
      421 atcgtcagcc ggtttgttat caccgaggct ttcttcctgg gcctgtgcaa tggtggccag
      481 tgcattttta gcacgctcgc gcagtgcttc aacggtcggc tcatcaaggc cttcgatttc
      541 caacagctct ttcatcggca cataggccaa ttcttccagc gtcgagaagc cttcttctac
      601 cagaacagtc gcgaagtctt cgtcgatgtc gagatatttg gtgaaggtgt cgatcgctgc
      661 gtgcgcttcc gcctgatgct tagcttgcag gtcgtcaacg gtcatcacgt tgagttccca
      721 accgctcagt tgcgaagcca gacgcacgtt ctgaccgtta cggccaatcg cctgcgccag
      781 attaccggct tcaacggcga tgtccatggt gtgtttatct tcatccacca cgatagaagc
      841 aacgtctgcc ggtgccattg cgttaatcac gaactgcgcc gggttatcat cccacaggac
      901 gatatcgata cgctcgccac ccagttcagt agacaccgcc tgaacacgcg cgccacgcat
      961 acctacgcaa gcacctaccg gatcgatacg tttatcgttg gttttcaccg cgattttcgc
     1021 acgagaaccc ggatcgcgag ccgctgcttt aatttcaatc acttcttcgc cgatttctgg
     1081 cacttcaata cggaacagtt cgatcagcat ttccggcttg gaacgagtga cgaacagttg
     1141 cgcgccacgc gcttccgggc gaacggaata gagcacgcca cgaacgcggt cgccagggcg
     1201 gaagttttca cgcggcagca tatcttcgcg caggatcacg gcttcagcgt tgttgcccag
     1261 atccagagag atgttgtcgc ggtttacttt tttcaccacg ccggtgatga tttcaccttc
     1321 gtgttcacgg aactgatcaa ccaccatcgc acgttcggct tcacgcactt tctgcacgat
     1381 aacctgtttt gccgtctggg tagtgatacg gtcaaaggta acagactcaa tctgatcttc
     1441 aacgtaatcg cccaggttca ggctttcatc ttcataacgt gcggcttcaa gggtgatttc
     1501 cttggtcggc tgggtgactt catcaacaac taaccagcga cggaaagtgt caaaatcacc
     1561 gcttttgcga tcgatctgta cgcggacgtc gatctcttgt tcatattttt tctttgttgc
     1621 tgtcgccagc gcgctttcca atgcttcgaa aatcttctcg cgaggtagcg ccttttcatt
     1681 ggatacggct tcaactacag ccaaaatttc tttgttgttg tgattatggt tgtggttgtg
     1741 gttatgatta tggttcggca tatgtatatc tccttcttaa agttaaacaa aattatttct
     1801 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
     1861 tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     1921 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     1981 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     2041 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     2101 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     2161 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     2221 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     2281 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     2341 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     2401 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     2461 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     2521 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     2581 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     2641 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     2701 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     2761 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     2821 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     2881 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     2941 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     3001 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     3061 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     3121 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     3181 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     3241 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     3301 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     3361 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     3421 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     3481 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     3541 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     3601 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     3661 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     3721 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     3781 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     3841 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3901 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3961 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     4021 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     4081 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     4141 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     4201 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     4261 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     4321 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     4381 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     4441 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     4501 gagagtgcac catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4561 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4621 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4681 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4741 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4801 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4861 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4921 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4981 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5041 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5101 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5161 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5221 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5281 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5341 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5401 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5461 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5521 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5581 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5641 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5701 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5761 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5821 attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5881 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5941 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6001 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6061 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6121 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6181 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6241 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6301 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6361 tgaatactca tactcttcct ttttcaaatt attgaagcat ttatcagggt tattgtctca
     6421 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6481 ttccccgaaa agtgccacct aaattgtaag cgttaatgtg aaccatcacc ctaatcaagt
     6541 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     6601 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     6661 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     6721 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
