The pAlterŪ-Ex1 Vector allows mutagenesis of the insert sequence and expression of the mutant directly from the vector without subcloning. The vector contains a novel polylinker which allows protein expression in vivo and in vitro. Selection of mutations in the cloned sequence is coupled to the selection for ampicillin resistance in the vector. 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 and in vitro expression. 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.

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LOCUS       CVU47102     5858 bp    DNA   circular  SYN       19-FEB-1996
DEFINITION  Cloning vector pALTER-Ex1, complete sequence.
ACCESSION   U47102
NID         g1197548
KEYWORDS    .
SOURCE      Cloning vector pALTER-Ex1.
  ORGANISM  Cloning vector pALTER-Ex1
            artificial sequence; cloning vectors.
REFERENCE   1  (bases 1 to 5858)
  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: 1197548
FEATURES               Location/Qualifiers
     source          1..5858
                     /organism="Cloning vector pALTER-Ex1"
     misc_feature    1
                     /note="T7 RNA polymerase transcription initiation site"
     misc_feature    5..77
                     /note="multiple cloning site"
     misc_feature    37..39
                     /note="T7 translation start codon"
     misc_feature    group(65..67,71..73)
                     /note="tac and SP6 translation start codons"
     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: 1197549"
                     /codon_start=1
                     /transl_table=11
                     /product="TetR"
                     /db_xref="PID:g1197549"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASH
                     YGVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAG
                     RIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLH
                     APFLAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTV
                     FFIMQLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGE
                     KQAIIAGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQG
                     QLQGSLAALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSR
                     ATST"
     misc_binding    801..828
                     /gene="tetR"
                     /bound_moiety="tetracycline repair oligonucleotide"
     CDS             complement(4212..4784)
                     /gene="ampR"
                     /note="NCBI gi: 1197550"
                     /codon_start=1
                     /function="ampicillin resistance"
                     /transl_table=11
                     /product="beta-lactamase"
                     /db_xref="PID:g1197550"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPQWQQRCANY"
     misc_binding    4232..4251
                     /gene="ampR"
                     /bound_moiety="ampicillin repair oligonucleotide"
     misc_feature    5223..5678
                     /note="phage f1 region"
     primer_bind     5794..5810
                     /note="pUC/M13 forward sequencing primer"
     promoter        join(5842..5858,1..6)
                     /note="T7 RNA polymerase promoter"
BASE COUNT     1363 a   1560 c   1553 g   1382 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 ttgttctcat 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 gaagtcagcg
     2281 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     2341 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     2401 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     2461 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     2521 agaaattccc ccttacacgg aggcatcaag tgaccaaaca ggaaaaaacc gcccttaaca
     2581 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
     2641 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
     2701 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     2761 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     2821 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     2881 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc ctatgcggtg
     2941 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     3001 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3061 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3121 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3181 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3241 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3301 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3361 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3421 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3481 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3541 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3601 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3661 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3721 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3781 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3841 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3901 tatatatgag taaactttgg ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3961 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4021 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4081 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4141 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4201 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gcggcatcgt ggtgtcacgc
     4261 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4321 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4381 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4441 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4501 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     4561 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4621 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4681 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4741 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4801 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4861 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4921 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4981 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     5041 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     5101 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5161 gtgcacccta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5221 cgaaattgta aacgttaata ttttgttaaa attcgcgtta aatatttgtt aaatcagctc
     5281 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5341 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5401 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5461 caaatcaagt tttttgcggt cgaggtgccg taaagctcta aatcggaacc ctaaagggag
     5521 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5581 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5641 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc
     5701 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     5761 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     5821 aaaacgacgg ccagtgaatt gtaatacgac tcactata
//