LOCUS       pSpeedET.        5464 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector with a lethal ccdB gene, for polymerase incomplete primer 
            extension (PIPE) cloning to express proteins with a cleavable 6xHis 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    pSpeedET
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5464)
  AUTHORS   Klock HE, Koesema EJ, Knuth MW, Lesley SA.
  TITLE     Combining the polymerase incomplete primer extension method for 
            cloning and mutagenesis with microscreening to accelerate structural
            genomics efforts.
  JOURNAL   Proteins 2008;71:982-94.
  PUBMED    18004753
REFERENCE   2  (bases 1 to 5464)
  AUTHORS   Joint Center for Structural Genomics (JCSG)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5464)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proteins"; 
            date: "2008"; volume: "71"; pages: "982-94"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     promoter        291..309
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             312..334
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             346..348
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             364..381
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             382..402
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     promoter        418..448
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             502..1158
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     CDS             1503..1805
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     terminator      2079..2165
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2257..2284
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        2304..2395
                     /label=AmpR promoter
     CDS             2396..3208
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      3382..3970
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4156..4296)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4563..5438)
                     /label=araC
                     /note="L-arabinose regulatory protein"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg taatacgact
      301 cactataggg ttttgtttaa ctttaagaag gagatataca tacccatggg atctgataaa
      361 attcatcatc atcatcatca cgaaaacctg tacttccagg gcgccggcac cccaggcttt
      421 acactttatg cttccggctc gtataatgtg tggattttga gttaggatcc ggcgagattt
      481 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
      541 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
      601 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
      661 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc
      721 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
      781 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
      841 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
      901 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
      961 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
     1021 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
     1081 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
     1141 gagtggcagg gcggggcgta aacgcgtgga tccggcttac taaaagccag ataacagtat
     1201 gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt
     1261 atgtcaaaaa gaggtgtgct atgaagcagc gtattacagt gacagttgac agcgacagct
     1321 atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca
     1381 gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc
     1441 tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca ggggctggtg
     1501 aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     1561 tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac
     1621 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     1681 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     1741 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     1801 gaatataact taattaactc tagataactg agaaggagat atacataccc gggtaagttt
     1861 aaacggtctc cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
     1921 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
     1981 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
     2041 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
     2101 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     2161 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
     2221 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
     2281 tttttgcgtt tctacaaact ctttttgttt atttttctaa atacattcaa atatgtatcc
     2341 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2401 ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga
     2461 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     2521 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     2581 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     2641 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     2701 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2761 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2821 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     2881 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2941 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     3001 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     3061 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     3121 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     3181 gtttcatttg atgctcgatg agtttttcta actgtcagac caagtttact catatatact
     3241 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3301 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3361 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3421 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3481 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3541 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3601 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3661 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3721 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3781 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3841 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3901 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3961 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4021 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4081 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4141 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4201 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4261 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4321 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4381 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag
     4441 atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat ggacgaagca
     4501 gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat
     4561 tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc
     4621 gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca
     4681 acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg
     4741 ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga
     4801 tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat atcaaaattg
     4861 ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt
     4921 ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc aagcagattt
     4981 atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac
     5041 aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt attggcaaat
     5101 attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg
     5161 tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc tggcgggaac
     5221 agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga
     5281 ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa
     5341 tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc attaaacgag
     5401 tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact cccgccattc
     5461 agag
//
