LOCUS       pMCSG8.        5340 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a 6xHis-S-loop-TEV leader for high-throughput 
            purification of toxic recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG8.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5340)
  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 5340)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5340)
  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..5340
                     /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(250..303)
                     /label=S loop
                     /note="GroES chaperone mobile loop that interacts with
                     GroEL"
     CDS             complement(322..339)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(340..342)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(350..372)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(387..411)
                     /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(412..430)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        743..820
                     /label=lacI promoter
     CDS             821..1900
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1916..1937
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2712..2900
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3005..3147
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3333..3921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4095..4952)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4953..5056)
                     /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 ctcggtacca gcagcagcag aaccggtcag aacgatacca ccagcagatt tggtttcaac
      301 ttccagatct acaccagaag aatgatgatg atgatggtgc atatgtatat ctccttctta
      361 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
      421 agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg
      481 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
      541 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
      601 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
      661 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
      721 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
      781 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
      841 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
      901 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
      961 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     1021 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     1081 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     1141 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     1201 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     1261 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     1321 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     1381 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     1441 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     1501 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     1561 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     1621 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     1681 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     1741 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     1801 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1861 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     1921 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     1981 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     2041 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     2101 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     2161 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     2221 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     2281 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     2341 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     2401 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     2461 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     2521 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     2581 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     2641 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     2701 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     2761 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     2821 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     2881 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     2941 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3001 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     3061 ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga
     3121 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     3181 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3241 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3301 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3361 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3421 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3481 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3541 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3601 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3661 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3721 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     3781 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3841 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3901 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3961 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4021 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4081 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4141 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4201 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4261 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4321 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4381 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     4441 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4501 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4561 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4621 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4681 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4741 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4801 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4861 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4921 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaaat tattgaagca
     4981 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5041 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatgt
     5101 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5161 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5221 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5281 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
