LOCUS pFLP-hyg 7055 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses FLP recombinase with hyg selection marker.
ACCESSION .
VERSION .
KEYWORDS pFLP-hyg
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7055)
AUTHORS Huang TW, Lam I, Chang HY, Tsai SF, Palsson BO, Charusanti P
TITLE Capsule deletion via a lambda-Red knockout system perturbs biofilm
formation and fimbriae expression in Klebsiella pneumoniae MGH
78578.
JOURNAL BMC Res Notes. 2014 Jan 8;7:13. doi: 10.1186/1756-0500-7-13.
PUBMED 24398052
REFERENCE 2 (bases 1 to 7055)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7055
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 10..555
/direction=RIGHT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind 456..475
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 672..689
/label=L4440
/note="L4440 vector, forward primer"
promoter 992..1096
/gene="bla"
/label=AmpR promoter
CDS 1097..2122
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(2311..2415)
/gene="bla"
/label=AmpR promoter
primer_bind 2483..2501
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
CDS complement(3449..4720)
/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="MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
YPAWNGIISQEVLDYLSSYINRRI"
RBS 4724..4732
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 4821..5534
/codon_start=1
/gene="cIts"
/product="temperature-sensitive variant of the phage lambda
repressor"
/label=-lambda repressor (ts)
/note="thermosensitivity is conferred by the A67T mutation"
/translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
VGKVIASQWPEETFG"
primer_bind 6041..6060
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 6125..6144
/label=pENTR-R
/note="pENTR vectors, reverse primer"
ORIGIN
1 atgatcttct tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac
61 cgccttgcag ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa
121 ctgccttgga ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca
181 tgacttcaag actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg
241 catgtctttc cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact
301 gaacgggggg ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg
361 tcaggcgtgg aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag
421 gcaggaacag gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt
481 cctgtcgggt ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg
541 cggagcctat ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta agtatcttcc
601 tggcatcttc caggaaatct ccgccccgtt cgtaagccat ttccgctcgc cgcagtcgaa
661 cgaccgagcg tagcgagtca gtgagcgagg aagcggaata tatcctgtat cacatattct
721 gctgacgcac cggtgcagcc ttttttctcc tgccacatga agcacttcac tgacaccctc
781 atcagtgcca acatagtaag ccagtataca ctccgctagc gctgatgtcc ggcggtgctt
841 ttgccgttac gcaccacccc gtcagtagct gaacaggagg gacagctgat agaaacagaa
901 gccactggag cacctcaaaa acaccatcat acactaaatc agtaagttgg cagcatcacc
961 cgacgcactt tgcagatctc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
1021 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
1081 ttgaaaaagg aagagtatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct
1141 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
1201 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
1261 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
1321 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
1381 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
1441 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
1501 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
1561 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
1621 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
1681 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
1741 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
1801 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
1861 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
1921 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
1981 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
2041 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
2101 tcgtccgagg gcaaaggaat agcgttcttc ggggcgaaaa ctctcaagga tcttaccgct
2161 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
2221 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
2281 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
2341 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
2401 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
2461 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt
2521 ttataaaccg tggagcgggc aatactgagc tgatgagcaa tttccgttgc accagtgccc
2581 ttctgatgaa gcgtcagcac gacgttcctg tccacggtac gcctgcggcc aaatttgatt
2641 cctttcagct ttgcttcctg tcggccctca ttcgtgcgct ctaggatcct ctacgccgga
2701 cgcatcgtgg ccggcatcac cggcgctgag gtctgcctcg tgaagaaggt gttgctgact
2761 cataccaggc ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga
2821 gagctttgtt gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt
2881 ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc
2941 aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
3001 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
3061 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
3121 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
3181 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
3241 agtgacgact gaatccggtg agaatggcag aataggaact tcggaatagg aacttcaaag
3301 cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg
3361 ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca
3421 tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt
3481 agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag
3541 cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca
3601 atgctatcat ttcctttgat attggatcat atgcatagta ccgagaaact agtgcgaagt
3661 agtgatcagg tattgctgtt atctgatgag tatacgttgt cctggccacg gcagaagcac
3721 gcttatcgct ccaatttccc acaacattag tcaactccgt taggcccttc attgaaagaa
3781 atgaggtcat caaatgtctt ccaatgtgag attttgggcc attttttata gcaaagattg
3841 aataaggcgc atttttcttc aaagctttat tgtacgatct gactaagtta tcttttaata
3901 attggtattc ctgtttattg cttgaagaat tgccggtcct atttactcgt tttaggactg
3961 gttcagaatt cctcaaaaat tcatccaaat atacaagtgg atcgatccta ccccttgcgc
4021 taaagaagta tatgtgccta ctaacgcttg tctttgtctc tgtcactaaa cactggatta
4081 ttactcccag atacttattt tggactaatt taaatgattt cggatcaacg ttcttaatat
4141 cgctgaatct tccacaattg atgaaagtag ctaggaagag gaattggtat aaagtttttg
4201 tttttgtaaa tctcgaagta tactcaaacg aatttagtat tttctcagtg atctcccaga
4261 tgctttcacc ctcacttaga agtgctttaa gcattttttt actgtggcta tttcccttat
4321 ctgcttcttc cgatgattcg aactgtaatt gcaaactact tacaatatca gtgatatcag
4381 attgatgttt ttgtccatag taaggaataa ttgtaaattc ccaagcagga atcaatttct
4441 ttaatgaggc ttccagaatt gttgcttttt gcgtcttgta tttaaactgg agtgatttat
4501 tgacaatatc gaaactcagc gaattgctta tgatagtatt atagctcatg aatgtggctc
4561 tcttgattgc tgttccgtta tgtgtaatca tccaacataa ataggttagt tcagcagcac
4621 ataatgctat tttctcacct gaaggtcttt caaacctttc cacaaactga cgaacaagca
4681 ccttaggtgg tgttttacat aatatatcaa attgtggcat acaacctcct tagtacatgc
4741 aaccattatc accgccagag gtaaaatagt caacacgcac ggtgttagat atttatccct
4801 tgcggtgata gatttaacgt atgagcacaa aaaagaaacc attaacacaa gagcagcttg
4861 aggacgcacg tcgccttaaa gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc
4921 aggaatctgt cgcagacaag atggggatgg ggcagtcagg cgttggtgct ttatttaatg
4981 gcatcaatgc attaaatgct tataacgccg cattgcttac aaaaattctc aaagttagcg
5041 ttgaagaatt tagcccttca atcgccagag aaatctacga gatgtatgaa gcggttagta
5101 tgcagccgtc acttagaagt gagtatgagt accctgtttt ttctcatgtt caggcaggga
5161 tgttctcacc taagcttaga acctttacca aaggtgatgc ggagagatgg gtaagcacaa
5221 ccaaaaaagc cagtgattct gcattctggc ttgaggttga aggtaattcc atgaccgcac
5281 caacaggctc caagccaagc tttcctgacg gaatgttaat tctcgttgac cctgagcagg
5341 ctgttgagcc aggtgatttc tgcatagcca gacttggggg tgatgagttt accttcaaga
5401 aactgatcag ggatagcggt caggtgtttt tacaaccact aaacccacag tacccaatga
5461 tcccatgcaa tgagagttgt tccgttgtgg ggaaagttat cgctagtcag tggcctgaag
5521 agacgtttgg ctgatcggca aggtgttctg gtcggcgcat agctgataac aattgagcaa
5581 gaatctgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
5641 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
5701 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
5761 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
5821 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
5881 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
5941 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
6001 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
6061 catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt
6121 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
6181 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc
6241 tttgttgaat aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca
6301 acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca
6361 aagctctcat caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcct
6421 ggtatgagtc agcaacacct tcttcacgag gcagacctca gcgccacagg tgcggttgct
6481 ggcgctaacc gtttttatca ggctctggga ggcagaataa atgatcatat cgtcaattat
6541 tacctccacg gggagagcct gagcaaactg gcctcaggca tttgagaagc acacggtcac
6601 actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg
6661 accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc attcatccgc
6721 ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata actgccttaa
6781 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
6841 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
6901 ttgtcgcctt gcgtataata tttgcccatg gtgcaccgat gataagctgt caaacatgag
6961 aattacaact tatatcgtat ggggctgact tcaggtgcta catttgaaga gataaattgc
7021 actgaaatct agaaatattt tatctgatta ataag
//
