LOCUS       pMCSG20.        5991 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with an S-Tag-GST-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG20
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5991)
  AUTHORS   Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID.
  TITLE     A family of LIC vectors for high-throughput cloning and purification
            of proteins.
  JOURNAL   Methods Mol. Biol. 2009;498:105-15.
  PUBMED    18988021
REFERENCE   2  (bases 1 to 5991)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5991)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods 
            Mol. Biol."; date: "2009"; volume: "498"; pages: "105-15"
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..5991
                     /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(271..924)
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
     CDS             complement(946..990)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(991..993)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1001..1023)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1038..1062)
                     /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(1063..1081)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1394..1471
                     /label=lacI promoter
     CDS             1472..2551
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2567..2588
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3363..3551
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3656..3798
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3984..4572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4746..5603)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5604..5707)
                     /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 agatctgggt aatcatccga ttttggagga tggtcgccac caccaaacgt
      301 ggcttgccag ccctgcaaag gccatgctat atacttgctg gatttcaagt acttatcaat
      361 ttgtgggata gcttcaatac gttttttaaa acaaactaat tttgggaacg catccaggca
      421 cattgggtcc atgtataaaa caacatcaag agcgtcatac aacatgaagt caggatgggt
      481 tacatgatca ccatttaaat atgttttatg acataaacga tcttcgaaca ttttcagcat
      541 ttcaggtagc ttgctaagaa aatcaacttt gagagtttca aagtctttac tatatgcaat
      601 tctcgaaaca ccgtatctaa tatccaaaac cgctccttca agcattgaaa tctctgcacg
      661 ctcttttgga caaccaccca acatgttgtg cttgtcagct atataacgta tgatggccat
      721 agactgtgtt aatttaacat caccatcaat ataataagga agattgggaa actccaaacc
      781 caattcaaac tttttgtttc gccatttatc accttcatcg cgctcataca aatgctcttc
      841 atatttttct tcaagatatt ccaaaagaag tcgagtgggt tgcacaaggc ccttaatttt
      901 ccaataacct agtatagggg acatcagatc tacgccggaa gatgggctgt ccatgtgctg
      961 gcgttcgaat ttagcagcag cggtttcttt catatgtata tctccttctt aaagttaaac
     1021 aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
     1081 aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt ggccggcatc
     1141 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
     1201 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
     1261 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
     1321 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
     1381 cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     1441 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     1501 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     1561 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     1621 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     1681 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     1741 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     1801 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     1861 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     1921 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     1981 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     2041 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     2101 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     2161 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     2221 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     2281 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     2341 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     2401 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     2461 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2521 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     2581 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
     2641 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     2701 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     2761 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     2821 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     2881 ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     2941 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     3001 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     3061 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     3121 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     3181 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     3241 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     3301 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     3361 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     3421 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     3481 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3541 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3601 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3661 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     3721 gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg
     3781 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3841 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3901 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3961 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4021 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4081 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4141 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4201 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4261 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4321 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4381 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4441 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4501 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4561 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4621 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4681 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4741 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4801 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4861 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4921 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4981 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5041 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5101 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5161 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5221 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5281 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5341 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5401 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5461 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5521 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5581 acacggaaat gttgaatact catactcttc ctttttcaaa ttattgaagc atttatcagg
     5641 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5701 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaatg tgaaccatca
     5761 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5821 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5881 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5941 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
