LOCUS       pMCSG66.        5396 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing a protein with a 6xHis-2xHA-TEV 
            leader plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG66
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5396)
  AUTHORS   Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, 
            Jedrzejczak R, Joachimiak A.
  TITLE     New LIC vectors for production of proteins from genes containing 
            rare codons.
  JOURNAL   J. Struct. Funct. Genomics 2013;14:135-44.
  PUBMED    24057978
REFERENCE   2  (bases 1 to 5396)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5396)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     There are two sites for ligation-independent cloning (LIC).
            To express a 6xHis-2xHA-TEV-tagged protein, linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
            To express a second untagged protein, linearize with SmaI and treat 
            with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5396
                     /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"
     RBS             243..248
                     /note="ribosome binding site"
     CDS             complement(279..299)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(306..332)
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             complement(333..359)
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             complement(378..395)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(396..398)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(406..428)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(443..467)
                     /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(468..486)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        799..876
                     /label=lacI promoter
     CDS             877..1956
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1972..1993
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2768..2956
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3061..3203
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3389..3977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4151..5008)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5009..5112)
                     /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 acttccaatg ttatggtggt gcccgggaga
      241 gactccttct taaagttaag ttatccactt ccaatattgg attggaagta caggttctcg
      301 gtacctgcgt agtccggtac gtcgtacgga tatgcgtagt ccggcacatc atacggatac
      361 agatctacac cagaagaatg atgatgatga tggtgcatat gtatatctcc ttcttaaagt
      421 taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc
      481 gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc atcgtggccg
      541 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
      601 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
      661 gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg
      721 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
      781 gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
      841 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
      901 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
      961 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     1021 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     1081 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     1141 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     1201 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     1261 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     1321 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     1381 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     1441 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     1501 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     1561 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     1621 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     1681 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     1741 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     1801 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     1861 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1921 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     1981 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     2041 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     2101 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     2161 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     2221 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     2281 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     2341 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     2401 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     2461 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     2521 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     2581 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     2641 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     2701 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     2761 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     2821 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     2881 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     2941 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     3001 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     3061 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     3121 tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg
     3181 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3241 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3301 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3361 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3421 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3481 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3541 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3601 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3661 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3721 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3781 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     3841 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3901 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3961 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4021 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4081 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4141 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4201 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4261 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4321 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4381 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4441 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4501 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4561 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4621 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4681 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4741 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4801 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4861 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4921 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4981 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaaattatt gaagcattta
     5041 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5101 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatgtgaac
     5161 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     5221 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     5281 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     5341 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
