pComb3X is the newest of the pComb vectors. Improvements over pComb3 include increased stability and introduction of an asymmetric SfiI cassette for directional cloning of full Fab, scFv, peptide and other protein for phage display. 6xHis and HA tags allow for purification and detection. An amber stop codon was introduced to turn-off expression of the pIII fusion protein by switching to a non-supressor strain of E. coli allowing production of soluble protein without subcloning. Alternatively, the gene for phage protein pIII can be removed by SpeI/NheI digest. pComb3XSS is recommended for preparation of vector for library cloning. The “SS” refers to the double stuffer, a 1200bp stuffer in the Fab light chain cloning region bounded by SacI and XbaI restriction sites and a 300bp stuffer in Fab heavy chain cloning region bound by XhoI and SpeI restriction sites. Also, the 1600bp double stuffer (both stuffers plus the leader sequence between the Fab light chain and heavy chain cloning regions) can be removed by SfiI digest so that non-Fab genes of interest can be cloned. Also available on Addgene: pComb3XTT and pComb3XLambda are only needed at templates for the construction of chimeric Fab libraries as described in Phage Display: A Laboratory Manual. pComb3XTT can also be used as an Fab expression control.
Summary
Bacterial Resistance(s): Ampicillin
Growth Temperature: 37°C
Growth Strain(s): TG1
Copy number: High Copy
Insert Size (bp): 1600
Promoter:lacZ
Tags / Fusion Proteins
6x His (C terminal on backbone)
HA tag (C terminal on backbone)
geneIII (C terminal on backbone)
5′ sequencing primer
5'-AAG ACA GCT ATC GCG ATT GCA G-3'
3′ sequencing primer
5'-GCC CCC TTA TTA GCG TTT GCC ATC-3'
Vector sequence
1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
121 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
181 aattgtgagc ggataacaat tgaattcagg aggaatttaa aatgaaaaag acagctatcg
241 cgattgcagt ggcactggct ggtttcgcta ccgtggccca ggcggccgag ctcgccatgg
301 ctggttgggc agcgagtaat aacaatccag cggctgccgt aggcaatagg tatttcatta
361 tgactgtctc cttggcgact agctagttta gaattcgtaa tcatggtcat agctgtttcc
421 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
481 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
541 cgctttccag tcgggaaacc tgtcgtgtta ctaatgatgg tgatggtgat ggctagtttt
601 gtcacaagat ttgggctcaa ctttcttgtc caccttggtg ttgctgggct tgtgattcac
661 gttgcagatg taggtctggg tgcccaagct gctggagggc acggtcacca cgctgctgag
721 ggagtagagt cctgaggact gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc
781 gcctgagttc cacgacaccg tcgccggttc ggggaagtag tccttgacca ggcagcccag
841 ggccgctgtg cccccagagg tgctcttgga ggagggtgcc agggggaaga ccgatgggcc
901 cttggtggag gctgcggaga cggtgaccgt ggtaccagca gaaacctggc caggctccca
961 ggctcctcat ctatggtaca tccagcaggg ccactggcat cccagacagg ttcagtggca
1021 gtgggtctgg gacagacttc actctcacca tcagcagact ggagcctgaa gattttgcag
1081 tgtactactg tcagcagtat ggtggctcac cgtggttcgg ccaagggacc aaggtggaac
1141 tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga
1201 aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag
1261 tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc
1321 aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc aaagcagact
1381 acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc ttgcccgtca
1441 caaagagctt caacagggga gagtgttagt tctagataat taattaggag gaatttaaaa
1501 tgaaatacct attgcctacg gcagccgctg gattgttatt actcgctgcc caaccagcca
1561 tggccgaggt gcagctgctc gagatgagcg ataaaattat tcacctgact gacgacagtt
1621 ttgacacgga tgtactcaaa gcggacgggg cgatcctcgt cgatttctgg gcagagtggt
1681 gcggtccgtg caaaatgatc gccccgattc tggatgaaat cgctgacgaa tatcagggca
1741 aactgaccgt tgcaaaactg aacatcgatc aaaaccctgg cactgcgccg aaatatggca
1801 tccgtggtat cccgactctg ctgctgttca aaaacggtga agtggcggca accaaagtgg
1861 gtgcactgtc taaaggtcag ttgaaagagt tcctcgacgc taacctggcg tacccgtacg
1921 acgttccgga ctacggttct actagtggcc aggccggcca gcaccatcac catcaccatg
1981 gcgcataccc gtacgacgtt ccggactacg cttcttagga gggtggtggc tctgagggtg
2041 gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg
2101 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg
2161 aaaacgtgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg
2221 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg
2281 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt
2341 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt
2401 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat
2461 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt
2521 ttgctaacat actgcgtaat aaggagtctt aagctagcta attaatttaa gcggccgcag
2581 atctgctctc tgaggaggat ctgggaaatt gtaagcgtta atattttgtt aaaattcgcg
2641 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
2701 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
2761 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
2821 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
2881 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
2941 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
3001 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
3061 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
3121 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
3181 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
3241 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
3301 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
3361 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
3421 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
3481 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
3541 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
3601 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
3661 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
3721 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
3781 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
3841 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
3901 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
3961 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
4021 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
4081 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
4141 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
4201 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
4261 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
4321 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
4381 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
4441 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
4501 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
4561 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
4621 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
4681 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
4741 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
4801 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
4861 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
4921 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
4981 gaggaagcgg a
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