一质粒简介：
酶切位点
BamHI（5791 bp）、PstI（4875 bp）、EcoRV（7482 bp）、NcoI（701 bp）、BsaAI（1313 bp）等。
pCP20的标准实验流程（抗性消除+质粒丢失）
1. 转化pCP20：将pCP20转化到含FRT侧翼抗性标记的大肠杆菌敲除株中，涂布于含氨苄青霉素的LB平板，30℃培养。
2. 诱导FLP表达：挑取单菌落，在42℃条件下震荡培养过夜（热诱导cI857失活，FLP大量表达，切除抗性标记）。
3. 质粒丢失：42℃同时抑制Rep101(Ts)功能，质粒无法复制，随细菌分裂逐渐丢失。
4. 验证：分别点种到含氨苄、含氯霉素和无抗平板上，若氨苄和氯霉素均敏感，说明抗性标记已消除且pCP20已丢失，得到无痕敲除菌株。
- 序列：
- GAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGT
- TCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAA
- CACCTTCTTCACGAGGCAGACCTCAGCGCCACAGGTGCGGTTGCTGGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTAT
- TACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATA
- AGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCA
- GGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCA
- TCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATA
- TTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCG
- TAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTG
- TAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCC
- GGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCA
- AAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAA
- AATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTA
- TCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAG
- TGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGG
- ACATCAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCTGTAGTGCCATTTACCCCCATTCACTGCCAGAGCCGTGAG
- CGCAGCGAACTGAATGTCACGAAAAAGACAGCGACTCAGGTGCCTGATGGTCGGAGACAAAAGGAATATTCAGCGATTTGCCCGAGCTTGCGAGGGTGCTACTTA
- AGCCTTTAGGGTTTTAAGGTCTGTTTTGTAGAGGAGCAAACAGCGTTTGCGACATCCTTTTGTAATACTGCGGAACTGACTAAAGTAGTGAGTTATACACAGGGC
- TGGGATCTATTCTTTTTATCTTTTTTTATTCTTTCTTTATTCTATAAATTATAACCACTTGAATATAAACAAAAAAAACACACAAAGGTCTAGCGGAATTTACAG
- AGGGTCTAGCAGAATTTACAAGTTTTCCAGCAAAGGTCTAGCAGAATTTACAGATACCCACAACTCAAAGGAAAAGGACTAGTAATTATCATTGACTAGCCCATC
- TCAATTGGTATAGTGATTAAAATCACCTAGACCAATTGAGATGTATGTCTGAATTAGTTGTTTTCAAAGCAAATGAACTAGCGATTAGTCGCTATGACTTAACGG
- AGCATGAAACCAAGCTAATTTTATGCTGTGTGGCACTACTCAACCCCACGATTGAAAACCCTACAAGGAAAGAACGGACGGTATCGTTCACTTATAACCAATACG
- TTCAGATGATGAACATCAGTAGGGAAAATGCTTATGGTGTATTAGCTAAAGCAACCAGAGAGCTGATGACGAGAACTGTGGAAATCAGGAATCCTTTGGTTAAAG
- GCTTTGAGATTTTCCAGTGGACAAACTATGCCAAGTTCTCAAGCGAAAAATTAGAATTAGTTTTTAGTGAAGAGATATTGCCTTATCTTTTCCAGTTAAAAAAAT
- TCATAAAATATAATCTGGAACATGTTAAGTCTTTTGAAAACAAATACTCTATGAGGATTTATGAGTGGTTATTAAAAGAACTAACACAAAAGAAAACTCACAAGG
- CAAATATAGAGATTAGCCTTGATGAATTTAAGTTCATGTTAATGCTTGAAAATAACTACCATGAGTTTAAAAGGCTTAACCAATGGGTTTTGAAACCAATAAGTA
- AAGATTTAAACACTTACAGCAATATGAAATTGGTGGTTGATAAGCGAGGCCGCCCGACTGATACGTTGATTTTCCAAGTTGAACTAGATAGACAAATGGATCTCG
- TAACCGAACTTGAGAACAACCAGATAAAAATGAATGGTGACAAAATACCAACAACCATTACATCAGATTCCTACCTACATAACGGACTAAGAAAAACACTACACG
- ATGCTTTAACTGCAAAAATTCAGCTCACCAGTTTTGAGGCAAAATTTTTGAGTGACATGCAAAGTAAGTATGATCTCAATGGTTCGTTCTCATGGCTCACGCAAA
- AACAACGAACCACACTAGAGAACATACTGGCTAAATACGGAAGGATCTGAGGTTCTTATGGCTCTTGTATCTATCAGTGAAGCATCAAGACTAACAAACAAAAGT
- AGAACAACTGTTCACCGTTACATATCAAAGGGAAAACTGTCCATATGCACAGATGAAAACGGTGTAAAAAAGATAGATACATCAGAGCTTTTACGAGTTTTTGGT
- GCATTTAAAGCTGTTCACCATGAACAGATCGACAATGTAACAGATGAACAGCATGTAACACCTAATAGAACAGGTGAAACCAGTAAAACAAAGCAACTAGAACAT
- GAAATTGAACACCTGAGACAACTTGTTACAGCTCAACAGTCACACATAGACAGCCTGAAACAGGCGATGCTGCTTATCGAATCAAAGCTGCCGACAACACGGGAG
- CCAGTGACGCCTCCCGTGGGGAAAAAATCATGGCAATTCTGGAAGAAATAGCGCCTGTTTCGTTTCAGGCAGGTTATCAGGGAGTGTCAGCGTCCTGCGGTTCTC
- CGGGGCGTTCGGGTCATGCAGCCCGTAATGGTGATTTACCAGCGTCTGCCAGGCATCAATTCTAGGCCTGTCTGCGCGGTCGTAGTACGGCTGGAGGCGTTTTCC
- GGTCTGTAGCTCCATGTTCGGAATGACAAAATTCAGCTCAAGCCGTCCCTTGTCCTGGTGCTCCACCCACAGGATGCTGTACTGATTTTTTTCGAGACCGGGCAT
- CAGTACACGCTCAAAGCTCGCCATCACTTTTTCACGTCCTCCCGGCGGCAGCTCCTTCTCCGCGAACGACAGAACACCGGACGTGTATTTCTTCGCAAATGGCGT
- GGCATCGATGAGTTCCCGGACTTCTTCCGGATTACCCTGAAGCACCGTTGCGCCTTCGCGGTTACGCTCCCTCCCCAGCAGGTAATCAACCGGACCACTGCCACC
- ACCTTTTCCCCTGGCATGAAATTTAACTATCATCCCGCGCCCCCTGTTCCCTGACAGCCAGACGCAGCCGGCGCAGCTCATCCCCGATGGCCATCAGTGCGGCCA
- CCACCTGAACCCGGTCACCGGAAGACCACTGCCCGCTGTTCACCTTACGGGCTGTCTGATTCAGGTTATTTCCGATGGCGGCCAGCTGACGCAGTAACGGCGGTG
- CCAGTGTCGGCAGTTTTCCGGAACGGGCAACCGGCTCCCCCAGGCAGACCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAGCGTTCAAGTAACCGGGCAT
- GTTCATCATCAGTAACCCGTATTGTGAGCATCCTCTCGCGTTTCATCGGTATCATTACCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACTAAACG
- GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT
- AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT
- GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA
- TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGT
- AGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGA
- TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG
- GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGG
- CGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTC
- TCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA
- ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTAT
- TGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATT
- ATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTTTATAAACCGTGGAGCGGGCAATACTGAGCTGATGAGCAATTT
- CCGTTGCACCAGTGCCCTTCTGATGAAGCGTCAGCACGACGTTCCTGTCCACGGTACGCCTGCGGCCAAATTTGATTCCTTTCAGCTTTGCTTCCTGTCGGCCCT
- CATTCGTGCGCTCTAGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTG
- AATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACG
- GTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAG
- TGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGC
- CGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATT
- AATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAGAATAGGAACTTCGGAATAGGAACTTCAA
- AGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATAT
- ACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCC
- ATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTC
- CTTTGATATTGGATCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCTGGCCACGGCAGAAGC
- ACGCTTATCGCTCCAATTTCCCACAACATTAGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATGTGAGATTTTGGGCCATT
- TTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTTTATTGTACGATCTGACTAAGTTATCTTTTAATAATTGGTATTCCTGTTTATTGCTTGAAGA
- ATTGCCGGTCCTATTTACTCGTTTTAGGACTGGTTCAGAATTCCTCAAAAATTCATCCAAATATACAAGTGGATCGATCCTACCCCTTGCGCTAAAGAAGTATAT
- GTGCCTACTAACGCTTGTCTTTGTCTCTGTCACTAAACACTGGATTATTACTCCCAGATACTTATTTTGGACTAATTTAAATGATTTCGGATCAACGTTCTTAAT
- ATCGCTGAATCTTCCACAATTGATGAAAGTAGCTAGGAAGAGGAATTGGTATAAAGTTTTTGTTTTTGTAAATCTCGAAGTATACTCAAACGAATTTAGTATTTT
- CTCAGTGATCTCCCAGATGCTTTCACCCTCACTTAGAAGTGCTTTAAGCATTTTTTTACTGTGGCTATTTCCCTTATCTGCTTCTTCCGATGATTCGAACTGTAA
- TTGCAAACTACTTACAATATCAGTGATATCAGATTGATGTTTTTGTCCATAGTAAGGAATAATTGTAAATTCCCAAGCAGGAATCAATTTCTTTAATGAGGCTTC
- CAGAATTGTTGCTTTTTGCGTCTTGTATTTAAACTGGAGTGATTTATTGACAATATCGAAACTCAGCGAATTGCTTATGATAGTATTATAGCTCATGAATGTGGC
- TCTCTTGATTGCTGTTCCGTTATGTGTAATCATCCAACATAAATAGGTTAGTTCAGCAGCACATAATGCTATTTTCTCACCTGAAGGTCTTTCAAACCTTTCCAC
- AAACTGACGAACAAGCACCTTAGGTGGTGTTTTACATAATATATCAAATTGTGGCATACAACCTCCTTAGTACATGCAACCATTATCACCGCCAGAGGTAAAATA
- GTCAACACGCACGGTGTTAGATATTTATCCCTTGCGGTGATAGATTTAACGTATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACGTCG
- CCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAA
- TGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGAT
- GTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTAC
- CAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAA
- GCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAA
- GAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGC
- TAGTCAGTGGCCTGAAGAGACGTTTGGCTGATCGGCAAGGTGTTCTGGTCGGCGCATAGCTGATAACAATTGAGCAAGAATCTGCATTTCTTTCCAGACTTGTTC
- AACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAA
- AGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC
- TGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCT
- GACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGA
- TTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCAT
- AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTT
