The following information pertains to libraries constructed in the pT7T3-Pac vector.
Briefly, cDNA synthesis was primed with a Not-dT18 primer to
allow for directional cloning. Double-stranded cDNAs were ligated
to Eco RI adapters, digested with Not I, and directionally cloned
into Not + Eco RI-digested pT7T3Pac vector. The exception is the
human fetal liver-spleen library, for which a Pac-dT18 primer was
used to prime first strand synthesis, and consequently after
ligation to Eco RI adapters the double-stranded cDNAs were
digested with Pac I (instead of Not I) and directionally cloned
into the Pac I and Eco RI sites of the Pac I + Eco RI digested
pT7T3Pac vector.
The pT7T3Pac vector is essentially the same as pT7T3 (Pharmacia)
except that it has a slightly different polylinker. Following is
the sequence of the pT7T3Pac polylinker and flanking sequences:
5'AGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGG CTCGTATGTTGTGTGGAATTGTG[AGCGGATAACAATTTCACACAGGA]AAC AGCTATGACATGATTACGAATT[TAATACGACTCACTATAGGGA]ATTTGGCC CTCGAGGCCAAGAATTCCCGACTACGTAGTCGGGGATCCGTCTTAATTAA GCGGCCGCAAGCTTAT[TCCCTTTAGTGAGGGTTAAT]TTTAGCTTGGCACT GGCCGTCGTTTTACAAC[GTCGTGACTGGGAAAAC] CCTGGCGTTACCCA ACTTAATCGCCTTGCAG3'.
Note that, in the pT7T3Pac vector, the M13 reverse sequencing primer [AGCGGATAACAATTTCACACAGGA] is located 50 nt upstream from the Sfi site and the M13 Universal Sequencing primer [GTTTTCCCAGTCACGAC] is located 53 nt downstream from the Hind III site. Furthermore, the T7 promoter primer [taatacgactcactataggga] is 4 nt upstream from the Sfi, and the T3 promoter primer [tccctttagtgagggttaat] is only 2 nucleotides downstream from the Hind III site. The vector is ampicillin resistant and it has an f1 origin for propagation in the form of single-stranded circles. The sequence of the Eco RI adapter is as follows: 5'OH AATTCGGCACGAGG 3'OH 3'OH GCCGTGCTCC 5'p
LOCUS pT7T3D-PAC 2897 bp dsDNA Circular
DEFINITION pT7T3-pac (directional cloning vector derived from pT7T3D)
FEATURES Location/Qualifiers
promoter 169..188
/label=T3 promoter
misc_feature 190..260
/label=MCS
promoter complement(263..283)
/label=T7 promoter
rep_origin 700
/label=pMB1_ori
CDS complement(1459..2319)
/label=ampR
ORIGIN
1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
121 ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagctaaaa ttaaccctca
181 ctaaagggaa taagcttgcg gccgcttaat taagacggat ccccgactac gtagtcggga
241 attcttggcc tcgagggcca aattccctat agtgagtcgt attaaattcg taatcatggt
301 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
361 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
421 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
481 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
541 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
601 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
661 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
721 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
781 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
841 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
901 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
961 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
1021 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
1081 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
1141 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
1201 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
1261 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
1321 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
1381 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
1441 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
1501 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
1561 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
1621 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
1681 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
1741 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
1801 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
1861 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
1921 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
1981 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
2041 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
2101 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
2161 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
2221 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
2281 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
2341 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
2401 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa
2461 acgttaatgt tttgttaaat ttcgcgttaa atatttgtta aatcagctta ttttttaacc
2521 agtaagcaga aaatgacaaa aatccttata aatcaaaaga atagaccgag ttagttgtga
2581 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtaaaac
2641 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt tttggaggtc
2701 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
2761 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
2821 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
2881 gccgctactg ggcgcgt
//
>pT7T3D-PAC cccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccag ggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctaaaattaaccctca ctaaagggaataagcttgcggccgcttaattaagacggatccccgactacgtagtcggga attcttggcctcgagggccaaattccctatagtgagtcgtattaaattcgtaatcatggt catagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccg gaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgt tgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcg gccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaa tacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagc aaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgccccc ctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgct cacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacg aaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacc cggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcga ggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaa gaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggta gctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc agattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctg acgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaagga tcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatg agtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct gtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacggg agggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctc cagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaa ctttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgc cagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgt cgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatccc ccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagt tggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgc catccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagt gtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacata gcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaagga tcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcag catcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaa aaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatatt attgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttaga aaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaa acgttaatgttttgttaaatttcgcgttaaatatttgttaaatcagcttattttttaacc agtaagcagaaaatgacaaaaatccttataaatcaaaagaatagaccgagttagttgtga gtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtaaaac cgtctatcagggcgatggcccactacgtgaaccatcacccaaatcaagtttttggaggtc gaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacg gggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctag ggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgc gccgctactgggcgcgt
The following is the sequence of pT7T3D-PAC (ampicillin resistant; 50ug/ml, host DH10B, V-type plasmid). Comment: EcoRI (GAATTC) & PacI (TTAATTAA) sites capitalized. Sequencing primers: T3, T7
tttaatacgactcactatagggaatttggccctcgaggccaaGAATTCccgactacgtag tcggggatccgtcTTAATTAAgcggccgcaagcttattccctttagtgagggttaatttt agcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaa cttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagag