LOCUS       pMCSG65.        5369 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing a protein with a 6xHis-HA-TEV leader
            plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG65.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5369)
  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 5369)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5369)
  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-HA-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..5369
                     /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(351..368)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(369..371)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(379..401)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(416..440)
                     /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(441..459)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        772..849
                     /label=lacI promoter
     CDS             850..1929
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1945..1966
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2741..2929
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3034..3176
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3362..3950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4124..4981)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4982..5085)
                     /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 agtccggcac atcatacgga tacagatcta caccagaaga atgatgatga
      361 tgatggtgca tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
      421 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga
      481 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
      541 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
      601 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
      661 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
      721 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
      781 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
      841 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
      901 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
      961 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     1021 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     1081 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     1141 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     1201 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     1261 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     1321 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     1381 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     1441 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     1501 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     1561 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     1621 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     1681 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     1741 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     1801 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     1861 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     1921 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     1981 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     2041 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     2101 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     2161 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     2221 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     2281 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     2341 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     2401 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     2461 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     2521 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     2581 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     2641 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     2701 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     2761 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     2821 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     2881 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     2941 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     3001 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     3061 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     3121 catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     3181 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3241 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3301 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3361 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3421 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3481 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3541 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3601 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3661 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3721 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3781 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3841 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3901 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3961 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4021 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4081 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4141 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4201 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4261 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4321 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4381 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     4441 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4501 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4561 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4621 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4681 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4741 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4801 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4861 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4921 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4981 tactcttcct ttttcaaatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5041 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5101 agtgccacct aaattgtaag cgttaatgtg aaccatcacc ctaatcaagt tttttggggt
     5161 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5221 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5281 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5341 cgccgctaca gggcgcgtcc cattcgcca
//
