The pAlterŪ-Ex2 Vector allows mutagenesis of the insert sequence and in vivo or in vitro expression of the mutant directly from the vector without subcloning. The vector has a p15a origin of replication which allows it to be maintained with colE1 vectors such as pBR322 and pUC. Selection of mutations in the cloned sequence is coupled to the selection for chloramphenicol resistance in the vector. Multiple rounds or mutagenesis can be performed by alternate selections of resistance to chloramphenicol or tetracycline. Transcription of the insert is controlled by opposing T7 and SP6 promoters. A tac promoter is provided in the same orientation as the SP6 promoter for in vivo expression. The T7 promoter can also be used for expression in vivo. A T7 transcription terminator is located outside of the multiple cloning sites to reduce transcription through the tetracycline gene. Ribosome binding sites are provided in both orientations and convenient cloning sites are provided for cloning at the ATG initiation codon. This vector is suitable for expression of amber supressor mutants in the INTERCHANGE(TM) System.

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LOCUS       CVU47103     5838 bp    DNA   circular  SYN       19-FEB-1996
DEFINITION  Cloning vector pALTER-Ex2, complete sequence.
ACCESSION   U47103
NID         g1197551
KEYWORDS    .
SOURCE      Cloning vector pALTER-Ex2.
  ORGANISM  Cloning vector pALTER-Ex2
            artificial sequence; cloning vectors.
REFERENCE   1  (bases 1 to 5838)
  AUTHORS   Lesley,S. and Bohnsack,R.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-JAN-1996) Melinda Brady, Production, Promega
            Corporation, 5445 East Cheryl Parkway, Madison, WI 53711, USA
COMMENT
            NCBI gi: 1197551
FEATURES               Location/Qualifiers
     source          1..5838
                     /organism="Cloning vector pALTER-Ex2"
     misc_feature    1
                     /note="T7 RNA polymerase transcription initiation site"
     misc_feature    5..77
                     /note="multiple cloning sites"
     misc_feature    37..39
                     /note="T7 translation start codon"
     misc_feature    71..73
                     /note="tac and SP6 translation start codon"
     misc_feature    109
                     /note="tac transcription initiation site"
     promoter        120..147
                     /note="tac promoter"
     promoter        172..194
                     /note="SP6 RNA polymerase promoter"
     misc_feature    177
                     /note="SP6 RNA polymerase transcription initiation site"
     primer_bind     212..228
                     /note="pUC/M13 reverse sequencing primer"
     CDS             719..1909
                     /gene="tetR"
                     /note="NCBI gi: 1197552"
                     /codon_start=1
                     /transl_table=11
                     /product="TetR"
                     /db_xref="PID:g1197552"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASH
                     YGVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAG
                     RIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLH
                     APFLAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTV
                     FFIMQLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGE
                     KQAIIAGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQG
                     QLQGSLAALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSR
                     ATST"
     misc_binding    801..828
                     /gene="tetR"
                     /bound_moiety="tetracycline repair oligonucleotide"
     CDS             complement(3361..3600)
                     /gene="cmR"
                     /note="NCBI gi: 1197553"
                     /codon_start=1
                     /function="chloramphenicol resistance"
                     /transl_table=11
                     /product="CmR"
                     /db_xref="PID:g1197553"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF
                     LKTVKKNKHKFYPAFIHILARLMNAHPVPYGNERR"
     misc_binding    3372..3396
                     /gene="cmR"
                     /bound_moiety="chloramphenicol repair oligonucleotide"
     misc_feature    5203..5658
                     /note="phage f1 region"
     primer_bind     5775..5795
                     /note="pUC/M13 forward sequencing primer"
     promoter        join(5822..5838,1..6)
                     /note="T7 RNA polymerase promoter"
BASE COUNT     1370 a   1537 c   1526 g   1405 t
ORIGIN      
        1 gggcgaattc gttaacttta agaaggagat atacccatgg aggcctctag actgcagcgg
       61 ccgcatgcat atggatcctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
      121 ttatacgagc cgatgattaa ttgtcaacag ggggatgggg agtaagcttg agtattctat
      181 agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      241 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      301 cctaatgagt gagctaacca cattaatttg cgttgcgctc actgcccgct ttccagtcgg
      361 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
      421 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      481 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      541 acgcaggaaa gaacatgaat taattctcat gtttgacagc ttatcatcgg ataacccctt
      601 ggggcctcta aacgggtctt gaggggtttt ttgttatcat gtttgacagc ttatcttcga
      661 ttagctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg caccgtgtat
      721 gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg ctgtaggcat
      781 aggcttggtt atgccggtac tgccgggcct cttgcgggat atcgtccatt ccgacagcat
      841 cgccagtcac tatggcgtgc tgctagcgct atatgcgttg atgcaatttc tatgcgcacc
      901 cgttctcgga gcactgtccg accgctttgg ccgccgccca gtcctgctcg cttcgctact
      961 tggagccact atcgactacg cgatcatggc gaccacaccc gtcctgtgga ttctctacgc
     1021 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     1081 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
     1141 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttacatgc
     1201 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     1261 gcaggagtcg cataagggag agcgccgacc gatgcccttg agagccttca acccagtcag
     1321 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     1381 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     1441 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     1501 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     1561 cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg
     1621 gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca
     1681 ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc
     1741 ggctcttacc agcctaactt cgatcactgg accgctgatc gtcacggcga tttatgccgc
     1801 ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg
     1861 cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg
     1921 cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga
     1981 actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc
     2041 agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg
     2101 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     2161 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     2221 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcccct
     2281 acgtgctgct gaagttgccc gcaacagaga gtggaaccaa ccggtgatac cacgatacta
     2341 tgactgagag tcaacgccat gagcggcctc atttcttatt ctgagttaca acagtccgca
     2401 ccgctgtccg gtagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     2461 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgcccaa cagtcccccg
     2521 gccacggggc ctgccaccat acccacgccg aaacaagcgc cctgcaccat tatgttccgg
     2581 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     2641 cgctaaccgt ttttatcagg ctctgggagg cagaataaat gatcatatcg tcaattatta
     2701 cctccacggg gagagcctga gcaaactggc ctcaggcatt tgagaagcac acggtcacac
     2761 tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac
     2821 cctgccctga accgacgacc gggtcgaatt tgctttcgaa tttctgccat tcatccgctt
     2881 attatcactt attcaggcgt agcaccaggc gtttaagggc accaataact gccttaaaaa
     2941 aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     3001 acatggaagc catcacagac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     3061 tcgccttgcg tataatattt gcccatcgtg aaaacggggg cgaagaagtt gtccatattg
     3121 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     3181 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     3241 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     3301 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     3361 tcaccgtctt tcattgccat acggaaccgg atgagcattc atcaggcggg caagaatgtg
     3421 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     3481 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     3541 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     3601 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
     3661 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
     3721 aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa
     3781 gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt
     3841 actgatttag tgtatgatgg tgtttttgag gtgctccagt ggcttctgtt tctatcagct
     3901 gtccctcctg ttcagctact gacggggtgg tgcgtaacgg caaaagcacc gccggacatc
     3961 agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt
     4021 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg
     4081 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     4141 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
     4201 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
     4261 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
     4321 cgtttcccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt
     4381 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg
     4441 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct
     4501 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag
     4561 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa
     4621 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag
     4681 ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag
     4741 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa
     4801 tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     4861 cgatataagt tgtaattctc atgtttgaca gcttatcatc taagaaacca ttattatcat
     4921 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     4981 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     5041 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     5101 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcacccta tgcggtgtga
     5161 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgaaattgta aacgttaata
     5221 ttttgttaaa attcgcgtta aatatttgtt aaatcagctc attttttaac caataggccg
     5281 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5341 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5401 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttgcggt
     5461 cgaggtgccg taaagctcta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5521 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5581 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5641 cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     5701 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     5761 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     5821 gtaatacgac tcactata
//