LOCUS       pEU-HSBC.        6336 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) Vector with the sacB gene, for in vitro transcription and 
            translation of an N-terminally 6xHis-tagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pEU-HSBC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6336)
  AUTHORS   Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL.
  TITLE     Cell-free protein production and labeling protocol for NMR-based 
            structural proteomics.
  JOURNAL   Nat. Methods 2004;1:149-53.
  PUBMED    15782178
REFERENCE   2  (bases 1 to 6336)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6336)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Methods"; date: "2004"; volume: "1"; pages: "149-53"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     To remove the sacB gene, insert an ORF between the SgfI site and the
            PmeI site or a downstream site.
FEATURES             Location/Qualifiers
     source          1..6336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        373..391
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             521..523
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             527..544
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             790..2208
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        2297..2327
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2381..3037
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    3050..3105
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3795..3811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3819..3835)
                     /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(3843..3873)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3888..3909)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4197..4785)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4959..5816)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5817..5921)
                     /label=AmpR promoter
ORIGIN
        1 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
       61 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
      121 cacgatgcgt ccggcgtaga ggatctggct agcgatgacc ctgctgattg gttcgctgac
      181 catttccggg tgcgggacgg cgttaccaga aactcagaag gttcgtccaa ccaaaccgac
      241 tctggcggca gtttacgaga gagatgatag ggtctgcttc agtaagccag atgctacaca
      301 attaggcttg tacatactgt cgttagaacg cggctacaat taatacataa ccttatgtat
      361 catacacata cgatttaggt gacactatag aatacacgga attcgcgttt ttatttttaa
      421 ttttctttca aatacttcta gctagagtat ttttacaaca attaccaaca acaacaacaa
      481 acaacaacaa cattacattt tacattctac aactacagcc atgggacatc atcatcatca
      541 tcacgcgatc gcccgttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa
      601 tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt
      661 atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg
      721 ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag
      781 acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc
      841 gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat
      901 aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag
      961 caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa aaatatctct
     1021 tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc
     1081 gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat
     1141 gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa
     1201 aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa
     1261 gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta
     1321 ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt
     1381 aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc
     1441 tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac
     1501 agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa
     1561 tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta
     1621 tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt
     1681 ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag
     1741 ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta
     1801 acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact
     1861 gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt
     1921 ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg
     1981 ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct
     2041 caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca
     2101 gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct
     2161 gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata aaaacgcaaa
     2221 agaaaatgcc gatatcctat tggcattttc ttttatttct tatcaacata aaggtgaata
     2281 gttaggcacc ccaggcttta cactttatgc tttcggctcg tataatgtgt ggattttgag
     2341 ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
     2401 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     2461 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     2521 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     2581 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
     2641 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     2701 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     2761 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
     2821 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     2881 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     2941 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     3001 attacaacag tactgcgatg agtggcaggg cggggcgtaa tgtttaaacg aattcgagct
     3061 cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagctgatcc ggctgctaac
     3121 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcaaaaaac
     3181 tagtttacgg ctagcgccgg atgcgacgcc ggtcgcgtct tatccggcct tcctatatca
     3241 ggctgtgttt aagacgccgc cgcttcgccc aaatccctat gccggttcga cggctggaca
     3301 aaatactgtt tatcttccca gcgcaggcag gttaatgtac caccccagca gcagccggta
     3361 tccagcgcgt atataccttc cggcgtacct ttgccctcca gcgatgccca gtgaccaaag
     3421 gcgatgctgt attcttcagc gacagggcca ggaatcgcaa accacggttt cagtggggca
     3481 ggggcctctt ccggcgattc ttactagcta gtatgcatag gtgctgaaat ataaagtttg
     3541 tgtttctaaa acacacgtgg tacgtacgat aacgtacagt gtttttccct ccacttaaat
     3601 cgaagggtag tgtcttggag cgcgcggagt aaacatatat ggttcatata tgtccgtagg
     3661 cacgtaaaaa aagcgaggga ttcgaattcc cccggaaccc ccggttgggg cccacgcctc
     3721 gatcgagcaa aaaaaaaaaa aaagaaaaaa aaaaaaaaaa aaagctttcc cgcggccagc
     3781 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3841 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3901 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3961 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4021 gcttcctcac tcactgactc gctgcgctcg gtcgctcggc tgcggcgagc ggtatcagct
     4081 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4141 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4201 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4261 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4321 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4381 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4441 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4501 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4561 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4621 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4681 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4741 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4801 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4861 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4921 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4981 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5041 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5101 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5161 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5221 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5281 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5341 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5401 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5461 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5521 tcattctgag aatagcgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5581 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5641 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5701 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5761 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5821 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5881 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5941 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6001 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     6061 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     6121 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     6181 attgtactga gagtgcacca ttcgacgctc tcccttatgc gactcctgca ttaggaagca
     6241 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6301 gatggcgccc aacagtcccc cggccacggg gcctgc
//
