LOCUS       40924_30131        5226 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding a C-terminal 6xHis tag, for 
            high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5226)
  AUTHORS   Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan
            K, Kim Y, Joachimiak A.
  TITLE     Cleavable C-terminal His-tag vectors for structure determination.
  JOURNAL   J. Struct. Funct. Genomics 2010;11:31-9.
  PUBMED    20213425
REFERENCE   2  (bases 1 to 5226)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5226)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2010"; volume: "11"; pages: "31-9"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5226
                     /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)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(208..225)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     RBS             complement(237..257)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(273..297)
                     /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(298..316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        629..706
                     /label=lacI promoter
     CDS             707..1786
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1802..1823
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2598..2786
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2891..3033
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3219..3807)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3981..4838)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4839..4942)
                     /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 tccgttaatg atgatgatgg tggtgcccgg gagagactcc
      241 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      301 atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc
      361 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
      421 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
      481 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
      541 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
      601 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
      661 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
      721 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
      781 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
      841 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
      901 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
      961 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     1021 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     1081 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     1141 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     1201 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     1261 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     1321 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     1381 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     1441 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     1501 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     1561 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     1621 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     1681 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     1741 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     1801 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     1861 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     1921 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     1981 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     2041 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     2101 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     2161 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     2221 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     2281 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     2341 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     2401 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     2461 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     2521 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     2581 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     2641 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     2701 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     2761 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     2821 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     2881 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     2941 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg
     3001 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3061 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3121 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3181 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3241 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3301 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3361 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3421 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3481 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3541 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3601 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3661 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3721 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3781 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3841 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3901 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3961 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4021 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4081 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4141 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4201 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4261 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     4321 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4381 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4441 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4501 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4561 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4621 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4681 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4741 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4801 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaaattatt
     4861 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4921 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     4981 taatgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5041 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5101 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5161 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     5221 tcgcca
//
