| 质粒名称: | pCMT-flp |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 67274 |
| 存储实验室: | Mark Liles |
|
载体骨架基本信息 |
|
| 载体名称: | pRham-flp-tetA |
| 载体出品公司: | Hossain el al (Mark Liles lab) |
| 载体抗性: | 四环素 |
| 空载体大小: | 3699 bp |
| 完整质粒大小: | 5813 bp |
| 载体类型: | Flp重组酶质粒，对温度敏感 |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 30 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | tetA, flp, repA101, oriR101 |
| 插入大小: | 5813 bp |
| 启动子 | tetA promoter of plasmid pACYC184 origin |
| 标签/融合蛋白: | His-SUMO (flp) （插入N端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | BsaAI（克隆过程中被破坏） |
| 3' 克隆位点: | AlwNI（在克隆过程中被破坏） |
| 5' 测序引物 | CGCATTCACAGTTCTCCGCAAG |
| 3' 测序引物 | CGAATCATCGGAAGAAGCAG |
|
文献引用方法 |
|
| 材料和方法部分: | pCMT-flp was a gift from Mark Liles (Addgene plasmid # 67274 ; http://n2t.net/addgene:67274 ; RRID:Addgene_67274) |
| 参考文献部分: | Genome modifications and cloning using a conjugally transferable recombineering system. Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR. Biotechnol Rep (Amst). 2015 Aug 28;8:24-35. doi: 10.1016/j.btre.2015.08.005. eCollection 2015 Dec. 10.1016/j.btre.2015.08.005 PubMed 28352570 |
买家导航
pCMTflp载体基本信息
pCMTflp载体图谱质粒图谱和多克隆位点信息
pCMTflp载体简介
pCMTflp载体序列
LOCUS pCMT-flp 5813 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Conjugally transferable Flp recombinase plasmid for unmarked
mutagenesis.
ACCESSION .
VERSION .
KEYWORDS pCMT-flp
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5813)
AUTHORS Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR
TITLE Genome modifications and cloning using a conjugally transferable
recombineering system.
JOURNAL Biotechnol Rep (Amst). 2015 Aug 28;8:24-35. doi:
10.1016/j.btre.2015.08.005. eCollection 2015 Dec.
PUBMED 28352570
REFERENCE 2 (bases 1 to 5813)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5813
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 48..79
/label=tonB terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"
rep_origin 641..863
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
CDS 911..1861
/codon_start=1
/gene="rep101 encoding an A56V mutation"
/product="temperature-sensitive version of the RepA protein
needed for replication with the pSC101 origin (Armstrong et
al., 1984)"
/label=Rep101(Ts)
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
terminator 2457..2486
/label=T3Te terminator
/note="phage T3 early transcription terminator"
terminator 2584..2631
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind 2627..2645
/label=T7 Term
/note="T7 terminator, reverse primer"
CDS complement(2660..3928)
/codon_start=1
/product="site-specific recombinase"
/label=FLP
/note="FLP is a site-specific recombinase from
Saccharomyces cerevisiae. Recombination occurs at FRT
sequences."
/translation="PQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMIT
HNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTII
PYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNS
FEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETK
TSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRS
YNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVART
TYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRY
PAWNGIISQEVLDYLSSYINRRI"
CDS complement(3929..4216)
/codon_start=1
/gene="S. cerevisiae SMT3 (truncated)"
/product="cleavable ubiquitin-like protein tag"
/label=SUMO
/translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
LMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
CDS complement(4229..4246)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
promoter 4340..4368
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
CDS 4416..5606
/codon_start=1
/gene="tet"
/product="tetracycline efflux protein"
/label=TcR
/note="confers resistance to tetracycline"
/translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
primer_bind complement(4768..4787)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 tcgctcaata ctgaccattt aaatcatacc tgacctccat agcagaaagt caaaagcctc
61 cgaccggagg cttttgactt gatcggcact aggtgaagta ggcccacccg cgagcgggtg
121 ttccttcttc actgtccctt attcgttcca ctgagcgtca gaccccgttg atgataccgc
181 tgccttactg ggtgcattag ccagtctgaa tgacctgtca cgggataatc cgaagtggtc
241 agactggaaa atcagagggc aggaactgct gaacagcaaa aagtcagata gcaccacata
301 gcagacccgc cataaaacgc cctgagaagc ccgtgacggg cttttcttgt attatgggta
361 gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca tttaccccca ttcactgcca
421 gagccgtgag cgcagcgaac tgaatgtcac gaaaaagaca gcgactcagg tgcctgatgg
481 tcggagacaa aaggaatatt cagcgatttg cccgattgcg gccgcaaccg agcttgcgag
541 ggtgctactt aagcctttag ggttttaagg tctgttttgt agaggagcaa acagcgtttg
601 cgacatcctt ttgtaatact gcggaactga ctaaagtagt gagttataca cagggctggg
661 atctattctt tttatctttt tttattcttt ctttattcta taaattataa ccacttgaat
721 ataaacaaaa aaaacacaca aaggtctagc ggaatttaca gagggtctag cagaatttac
781 aagttttcca gcaaaggtct agcagaattt acagataccc acaactcaaa ggaaaaggac
841 tagtaattat cattgactag cccatctcaa ttggtatagt gattaaaatc acctagacca
901 attgagatgt atgtctgaat tagttgtttt caaagcaaat gaactagcga ttagtcgcta
961 tgacttaacg gagcatgaaa ccaagctaat tttatgctgt gtggcactac tcaaccccac
1021 gattgaaaac cctacaagga aagaacggac ggtatcgttc acttataacc aatacgttca
1081 gatgatgaac atcagtaggg aaaatgctta tggtgtatta gctaaagcaa ccagagagct
1141 gatgacgaga actgtggaaa tcaggaatcc tttggttaaa ggctttgaga ttttccagtg
1201 gacaaactat gccaagttct caagcgaaaa attagaatta gtttttagtg aagagatatt
1261 gccttatctt ttccagttaa aaaaattcat aaaatataat ctggaacatg ttaagtcttt
1321 tgaaaacaaa tactctatga ggatttatga gtggttatta aaagaactaa cacaaaagaa
1381 aactcacaag gcaaatatag agattagcct tgatgaattt aagttcatgt taatgcttga
1441 aaataactac catgagttta aaaggcttaa ccaatgggtt ttgaaaccaa taagtaaaga
1501 tttaaacact tacagcaata tgaaattggt ggttgataag cgaggccgcc cgactgatac
1561 gttgattttc caagttgaac tagatagaca aatggatctc gtaaccgaac ttgagaacaa
1621 ccagataaaa atgaatggtg acaaaatacc aacaaccatt acatcagatt cctacctaca
1681 taacggacta agaaaaacac tacacgatgc tttaactgca aaaattcagc tcaccagttt
1741 tgaggcaaaa tttttgagtg acatgcaaag taagtatgat ctcaatggtt cgttctcatg
1801 gctcacgcaa aaacaacgaa ccacactaga gaacatactg gctaaatacg gaaggatctg
1861 aggttcttat ggctcttgta tctatcagtg aagcatcaag actaacaaac aaaagtagaa
1921 caactgttca ccgttacata tcaaagggaa aactgtccat acccatgatc taatgaatcg
1981 gccaacgcga accccttgcg gccgcccggg ccgtcgacca attctcatgt ttgacagctt
2041 atcatcgaat ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc
2101 gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag
2161 tactgttgta attcattaag cattctgccg acatggaagc catctggtaa caggattagc
2221 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
2281 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctc ggaaaaagag
2341 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
2401 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatt ttctaccgaa
2461 gaaaggccca cccgtgaagg tgagccagtg agttgattgc agtccagtta cgctggagtc
2521 tgaggctcgt cctgaatgat atcaagcttg aattcgttac ccggatatag ttcctccttt
2581 cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc
2641 agcggtggcg gccgctctat tatatgcgtc tatttatgta ggatgaaagg tagtctagta
2701 cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct
2761 ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca
2821 tttcctttga tattggatca tatgcatagt accgagaaac tagtgcgaag tagtgatcag
2881 gtattgctgt tatctgatga gtatacgttg tcctggccac ggcagaagca cgcttatcgc
2941 tccaatttcc cacaacatta gtcaactccg ttaggccctt cattgaaaga aatgaggtca
3001 tcaaatgtct tccaatgtga gattttgggc cattttttat agcaaagatt gaataaggcg
3061 catttttctt caaagcttta ttgtacgatc tgactaagtt atcttttaat aattggtatt
3121 cctgtttatt gcttgaagaa ttgccggtcc tatttactcg ttttaggact ggttcagaat
3181 tcctcaaaaa ttcatccaaa tatacaagtg gatcgatcct accccttgcg ctaaagaagt
3241 atatgtgcct actaacgctt gtctttgtct ctgtcactaa acactggatt attactccca
3301 gatacttatt ttggactaat ttaaatgatt tcggatcaac gttcttaata tcgctgaatc
3361 ttccacaatt gatgaaagta gctaggaaga ggaattggta taaagttttt gtttttgtaa
3421 atctcgaagt atactcaaac gaatttagta ttttctcagt gatctcccag atgctttcac
3481 cctcacttag aagtgcttta agcatttttt tactgtggct atttccctta tctgcttctt
3541 ccgatgattc gaactgtaat tgcaaactac ttacaatatc agtgatatca gattgatgtt
3601 tttgtccata gtaaggaata attgtaaatt cccaagcagg aatcaatttc tttaatgagg
3661 cttccagaat tgttgctttt tgcgtcttgt atttaaactg gagtgattta ttgacaatat
3721 cgaaactcag cgaattgctt atgatagtat tatagctcat gaatgtggct ctcttgattg
3781 ctgttccgtt atgtgtaatc atccaacata aataggttag ttcagcagca cataatgcta
3841 ttttctcacc tgaaggtctt tcaaaccttt ccacaaactg acgaacaagc accttaggtg
3901 gtgttttaca taatatacca aattgtggac ctccaatctg ttcgcggtga gcctcaataa
3961 tatcgttatc ctccatgtcc aaatcttcag gggtctgatc agcttgaatt tcaataccgt
4021 cgtacaagaa cgttaaggag tccatttcct taccctgtct tttagcgaac gcttccatca
4081 gccttcttaa aggagtggtc tttttgatct tgaagaagat ctctgaagat ccatcggaca
4141 cctttaaatt gatgtgagtc tcaggcttga cttctggctt gacctctggc ttagcttctt
4201 gattgacttc tgagtcctgc agggacccgt gatggtggtg atgatgcata tgtatatctc
4261 cttcttatag ttaaacaaaa ttatttctag tgtagcacct gaagtcagcc ccatacgata
4321 taagttgtaa ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat
4381 cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg
4441 tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc
4501 cgggcctctt gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc
4561 tagcgctata tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc
4621 gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga
4681 tcatggcgac cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca
4741 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
4801 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
4861 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
4921 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
4981 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
5041 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
5101 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
5161 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
5221 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
5281 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
5341 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
5401 atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga
5461 tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt
5521 tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
5581 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
5641 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aacccaaccc
5701 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
5761 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgctag ata
//
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