LOCUS       sequence-177297-        7055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLP recombinase with hyg selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Res 
            Notes."; date: "2014-01-8"; volume: "7:13. doi"; pages: " 
            10.1186/1756-0500-7-13"
COMMENT     Sequence Label: pFLP-hyg
FEATURES             Location/Qualifiers
     source          1..7055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     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
//
