LOCUS       pMCSG18.        6009 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a 6xHis-TEV leader for high-throughput 
            purification of C-terminally GFP-tagged proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG18.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6009)
  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 6009)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6009)
  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..6009
                     /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(214..921)
                     /label=GFP
                     /note="Aequorea victoria green fluorescent protein"
     CDS             complement(946..966)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(991..1008)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1009..1011)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1019..1041)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1056..1080)
                     /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(1081..1099)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1412..1489
                     /label=lacI promoter
     CDS             1490..2569
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2585..2606
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3381..3569
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3674..3816
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4002..4590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4764..5621)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5622..5725)
                     /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 tccgttatca gttgtacagt tcatccatgc catgtgtaat
      241 cccagcagct gttacaaact caagaaggac catgtggtct ctcttttcgt tgggatcttt
      301 cgaaagggca gattgtgtgg acaggtaatg gttgtctggt aaaaggacag ggccatcgcc
      361 aattggagta ttttgttgat aatggtctgc tagttgaacg cttccatctt caatgttgtg
      421 gcgggtcttg aagttcactt tgattccatt cttttgtttg tctgccatga tgtatacatt
      481 gtgtgagtta tagttgtatt ccaatttgtg tcccagaatg ttgccatctt ccttgaagtc
      541 aatacctttt aactcgattc tattaacaag ggtatcacct tcaaacttga cttcagcacg
      601 tgtcttgtag ttgccgtcat ctttgaagaa gatggtcctt tcctgtacat aaccttcggg
      661 catggcactc ttgaaaaagt catgccgttt catatgatcc gggtatcttg aaaagcattg
      721 aacaccatag cacagagtag tgactagtgt tggccatgga acaggcagtt tgccagtagt
      781 gcagatgaac ttcagggtaa gttttccgta tgttgcatca ccttcaccct ctccactgac
      841 agagaacttg tggccgttaa catcaccatc taattcaaca agaattggga caactccagt
      901 gaagagttct tctcctttgc tagccagtaa tccacttcca atattggatt ggaagtacag
      961 gttctcggta cccagatcta caccagaaga atgatgatga tgatggtgca tatgtatatc
     1021 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     1081 cctatagtga gtcgtattaa tttcgcggga tcgagatcga tctcgatcct ctacgccgga
     1141 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     1201 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     1261 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     1321 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     1381 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     1441 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1501 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1561 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     1621 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     1681 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     1741 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     1801 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     1861 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     1921 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     1981 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     2041 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     2101 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     2161 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     2221 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     2281 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     2341 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     2401 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2461 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2521 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     2581 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     2641 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2701 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     2761 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     2821 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     2881 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     2941 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     3001 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     3061 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     3121 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     3181 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     3241 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     3301 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     3361 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     3421 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     3481 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     3541 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3601 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3661 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     3721 taactatgcg gcatcagagc agattgtact gagagtgcac catatatgcg gtgtgaaata
     3781 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3841 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3901 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3961 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4021 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4081 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4141 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4201 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4261 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4321 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4381 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4441 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4501 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4561 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4621 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4681 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4741 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4801 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4861 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4921 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4981 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5041 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     5101 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5161 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5221 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5281 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5341 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5401 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5461 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5521 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5581 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaaatt
     5641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     5761 cgttaatgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5821 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     5881 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     5941 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6001 cattcgcca
//
