OGEM®-T Vector (plasmid) Sequence (credit: Promega Corporation, Madison, WI) Xmnl 1994 Scal 1875 Amp PGEM®-T Vector (300

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Ogem T Vector Plasmid Sequence Credit Promega Corporation Madison Wi Xmnl 1994 Scal 1875 Amp Pgem T Vector 300 1 (137.67 KiB) Viewed 13 times

The sequence supplied below is that of the pGEM(R)-T plasmid.The pGEM(R)-T plasmid was linearized with EcoR V (GATATC)at base 51(*), and this is the site where genes of interest (SB##) orcontrols (Luciferase, PC2) got inserted and ligated.
1 GGGCGAATTG GGCCCGACGT CGCATGCTCC CGGCCGCCATGGCCGCGGGA
51 T*ATCACTAGT GCGGCCGCCT GCAGGTCGAC CATATGGGAGAGCTCCCAAC
101 GCGTTGGATG CATAGCTTGA GTATTCTATA GTGTCACCTAAATAGCTTGG
151 CGTAATCATG GTCATAGCTG TTTCCTGTGT GAAATTGTTATCCGCTCACA
201 ATTCCACACA ACATACGAGC CGGAAGCATA AAGTGTAAAGCCTGGGGTGC
251 CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCACTGCCCGCTT
301 TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAATCGGCCAACGC
351 GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTTCCTCGCTCAC
401 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTATCAGCTCACT
451 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAACGCAGGAAAG
501 AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTAAAAAGGCCGC
551 GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAGCATCACAAAA
601 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACTATAAAGATAC
651 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTGTTCCGACCCT
701 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGAAGCGTGGCGC
751 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTAGGTCGTTCGC
801 TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCGACCGCTGCGC
851 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGACACGACTTAT
901 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGCGAGGTATGTA
951 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACGGCTACACTAG
1001 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTTACCTTCGGAA
1051 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGCTGGTAGCGGT
1101 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAAAAGGATCTCA
1151 AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAGTGGAACGAAA
1201 ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAGGATCTTCACC
1251 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCTAAAGTATATA
1301 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGTGAGGCACCTA
1351 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTGACTCCCCGTC
1401 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCCCCAGTGCTGC
1451 AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTATCAGCAATAA
1501 ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGCAACTTTATCC
1551 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAGTAAGTAGTTC
1601 GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACAGGCATCGTGG
1651 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGGTTCCCAACGA
1701 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAGCGGTTAGCTC
1751 CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCAGTGTTATCAC
1801 TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCATGCCATCCGTA
1851 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCATTCTGAGAATA
1901 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATACGGGATAATA
1951 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGGAAAACGTTCT
2001 TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGATCCAGTTCGAT
2051 GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTTACTTTCACCA
2101 GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGCAAAAAAGGGA
2151 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCCTTTTTCAATA
2201 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGATACATATTTG
2251 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCACATTTCCCCGA
2301 AAAGTGCCAC CTGATGCGGT GTGAAATACC GCACAGATGCGTAAGGAGAA
2351 AATACCGCAT CAGGAAATTG TAAGCGTTAA TATTTTGTTAAAATTCGCGT
2401 TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGCCGAAATCGGC
2451 AAAATCCCTT ATAAATCAAA AGAATAGACC GAGATAGGGTTGAGTGTTGT
2501 TCCAGTTTGG AACAAGAGTC CACTATTAAA GAACGTGGACTCCAACGTCA
2551 AAGGGCGAAA AACCGTCTAT CAGGGCGATG GCCCACTACGTGAACCATCA
2601 CCCTAATCAA GTTTTTTGGG GTCGAGGTGC CGTAAAGCACTAAATCGGAA
2651 CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG ACGGGGAAAGCCGGCGAACG
2701 TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGCTAGGGCGCTG
2751 GCAAGTGTAG CGGTCACGCT GCGCGTAACC ACCACACCCGCCGCGCTTAA
2801 TGCGCCGCTA CAGGGCGCGT CCATTCGCCA TTCAGGCTGCGCAACTGTTG
2851 GGAAGGGCGA TCGGTGCGGG CCTCTTCGCT ATTACGCCAGCTGGCGAAAG
2901 GGGGATGTGC TGCAAGGCGA TTAAGTTGGG TAACGCCAGGGTTTTCCCAG
2951 TCACGACGTT GTAAAACGAC GGCCAGTGAA TTGTAATACGACTCACTATA
Practice questions:
1) Find the EcoRV sequence.
2) Design primers to initiate DNA synthesis fivenucleotides downstream and upstream from the EcoRV site, whichwould yield a product that includes our genes ofinterest.
3) Design primers to initiate DNA synthesis fivenucleotides downstream and upstream from the EcoRV site, whichwould yield a product that does NOT include our genes of interest,but instead includes the remainder of pGEM sequence.
4) Find the following promoter sequences (these may notbe in the orientation shown above)?
T7 Promoter:TAATACGACTCACTATAGGG
SP6Promoter:ATTTAGGTGACACTATAG
OGEM®-T Vector (plasmid) Sequence (credit: Promega Corporation, Madison, WI) Xmnl 1994 Scal 1875 Amp PGEM®-T Vector (3000bp) ori f1 ori lacZ Nael 2692 T7 ↓ lac operator beta-lactamase (AmpR) coding region T7 RNA polymerase promoter (-17 to +3) Apal Aatll Sphl BstZl Ncol Sacll Spel Notl BstZl Pstl Sall Ndel Sacl BstXI Nsil † SP6 1 start 14 20 26 31 37 46 55 62 62 73 75 82 94 103 112 126 0356VA04 PGEM®-T Vector reference points: Base pairs T7 RNA polymerase transcription initiation site multiple cloning region/site SP6 RNA polymerase promoter (-17 to +3) SP6 RNA polymerase transcription initiation site lacz start codon 3000 1 10-113 124-143 126 165 185-201 1322-2182 2984-3