LOCUS       pMCSG62.        5869 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding tRNA genes for rare Arg and Ile codons, 
            for expressing a protein with a 6xHis-AviTag(TM)-TEV leader plus the
            BirA biotin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG62.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5869)
  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 5869)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5869)
  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     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..5869
                     /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(244..1206)
                     /label=BirA
                     /note="E. coli biotin protein ligase"
     RBS             1215..1220
                     /note="ribosome binding site"
     CDS             complement(1251..1271)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(1278..1322)
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             complement(1329..1346)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1347..1349)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1357..1379)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1394..1418)
                     /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(1419..1437)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1712..1788)
                     /label=argU
     tRNA            complement(2058..2133)
                     /label=ileX
     promoter        2273..2350
                     /label=lacI promoter
     CDS             2351..3430
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3446..3467
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3862..4450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4624..5481)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5482..5585)
                     /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 gcccagctta
      241 ttatttttct gcactacgca gggatatttc accgcccatc cagggtttta ttattccatc
      301 ctgctcaagt aataaagccc cctgtttgtc tattccgcgt gaaatgccaa atatctcttt
      361 atcaccaatg ataagtttca ctgggcgatt aataaaatta tccagctttt cccagcgcga
      421 cagataaggt gccaatcctt cttgttcgaa gagttccaac gcagcacgta attcacgtat
      481 tagcatggcc gccaacgtat tacgatcgag attgatcccc gcttcctgca gcgtgatcca
      541 cccctgatta acgacactct cttcaacacg gcgcattgcc atgttgatcc cggctccaat
      601 gactatttgc gccgcatcgc cagttttgcc agtcagctcc acaagaatgc ctgccagctt
      661 gcgatcctgc agatagaggt cattaggcca tttgacacga actttatctg ctcccagctt
      721 gcgtaatact tccgccatca cgataccgat aaccagactt aaaccaatcg ccgccgccgg
      781 gccttgttcc agacgccaga acatcgacaa atataagttt gcgccaaaag gcgaaaacca
      841 tttccgcccc cggcgaccac ggccagcctg ctggtattct gcaacacagg catcgcccga
      901 tttaagctct ccgatacgat caagaaggta ctgattcgtg gagtcaataa ctggcagcac
      961 ggctacacta ccgccatcca gctgacccaa tatctgttca gcattaagta actggatggg
     1021 ctcaggcagg ctgtatcctt tacccggaac ggtaaagaca tcaacgcccc agtcacgcag
     1081 tgtctgaatg tgtttattaa tagccgcccg gctcattccc agcgtttcac ccaactgctc
     1141 gccagagtga aattcaccgt tcgctaacag ggcaatcaat ttcagtggca cggtgttatc
     1201 cttcatggga gagactcctt cttaaagtta agttatccac ttccaatatt ggattggaag
     1261 tacaggttct cggtaccttc atgccactca attttttgcg cctcaaaaat atcgttcagt
     1321 ccagaagaat gatgatgatg atggtgcata tgtatatctc cttcttaaag ttaaacaaaa
     1381 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt
     1441 tcgcgggatc gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     1501 gtggattcag tcaggcgtcc cattatcagt gcttcaggaa atgggcggat gggagtccat
     1561 agaaatggtt cgtaggtatg ctcaccttgc gcctaatcat ttgacagagc atgcgaggaa
     1621 aatagacgac atttttggtg ataatgtccc aaatatgtcc cactctggaa ttatggagga
     1681 tataaagaag gcgtaactga ttgaattgta atggcgcgcc ctgcaggatt cgaacctgcg
     1741 gcccacgact tagaaggtcg ttgctctatc caactgagct aagggcgcgt tgataccgca
     1801 atgcggtgta atcgcgtgaa ttatacggtc aacccttgct gagtcaatgg cttttgatct
     1861 cgccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
     1921 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
     1981 cgtggccggg ggactgttgg gcgccatctc cttgcatgca aaaaggctga cgatttctcg
     2041 tcagcctttg ctatatctgg tggcccctgc tggacttgaa ccagcgacca agcgattatg
     2101 agtcgcctgc tctaaccact gagctaaggg gccatggtag cggattataa agtaactccg
     2161 tgtcgcaatc cgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     2221 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     2281 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     2341 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     2401 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     2461 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     2521 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     2581 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     2641 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     2701 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     2761 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     2821 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     2881 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     2941 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     3001 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     3061 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     3121 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     3181 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     3241 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     3301 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     3361 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     3421 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     3481 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     3541 tgactacagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     3601 agattgtact gagagtgcac catatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     3661 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3721 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3781 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3841 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3901 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3961 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4021 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4081 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4141 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4201 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4261 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4321 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4381 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4441 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4501 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4561 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4621 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4681 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4741 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4801 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4861 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4921 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4981 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5041 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5101 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5161 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5221 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5281 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5341 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5401 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5461 acggaaatgt tgaatactca tactcttcct ttttcaaatt attgaagcat ttatcagggt
     5521 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5581 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatgtg aaccatcacc
     5641 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5701 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5761 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5821 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
