LOCUS       sequence-240914-        5252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a mock vector for generating FLIP-FlpE targeting vectors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5252)
  AUTHORS   Andersson-Rolf A, Mustata RC, Merenda A, Kim J, Perera S, Grego T, 
            Andrews K, Tremble K, Silva JC, Fink J, Skarnes WC, Koo BK
  TITLE     One-step generation of conditional and reversible gene knockouts.
  JOURNAL   Nat Methods. 2017 Jan 30. doi: 10.1038/nmeth.4156.
  PUBMED    28135257
REFERENCE   2  (bases 1 to 5252)
  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: "Nat 
            Methods. 2017 Jan 30. doi: 10.1038/nmeth.4156."
COMMENT     Sequence Label: pUC118-FLIP-FlpE-Puro
FEATURES             Location/Qualifiers
     source          1..5252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    232..253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(268..273,274..291,292..298)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..322
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     311..333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     330..346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(452..485)
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(532..565)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    complement(532..565)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(572..605)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(625..644)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     679..698
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(968..1567)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1051..1071)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1548..1567
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    complement(1696..1729)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    1735..1768
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        complement(1779..2278)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1827..1846)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     2220..2243
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    2286..2319
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    2286..2319
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(2506..2523)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2506..2522)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2515..2537)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2735..3190
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2822..2841)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3032..3053
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3207..3226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3326..3348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3386..3404)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3472..3576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3577..3645,3646..4437)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3795..3814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4608..5196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5097..5116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgaatt
      361 cgagctcggt acccggggat cctctagagt cgtgtgaaga gcgcgatcgc gtttaaacgc
      421 tcttcagtaa gtatcaaaga tctggcgcgc cataacttcg tatagtacac attatacgaa
      481 gttatggcta agctttgtgc catgccattg atgctagcac agcctgaatg gataacttcg
      541 tataggatac cttatacgaa gttatgtcga cgaagttcct atactatcta gagaatagga
      601 acttcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
      661 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
      721 tgcaattaac aagtgccata gagcccaccg catccccagc atgcctgcta ttgtcttccc
      781 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca
      841 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca
      901 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacat
      961 caggaattca ggcaccgggc ttgcgggtca tgcaccaggt gcgcggtcct tcgggcacct
     1021 cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa ggggaggttg cggggcgcgg
     1081 aggtctccag gaaggcgggc accccggcgc gctcggccgc ctccactccg gggagcacga
     1141 cggcgctgcc cagacccttg ccctggtggt cgggcgagac gccgacggtg gccaggaacc
     1201 acgcgggctc cttgggccgg tgcggcgcca ggaggccttc catctgttgc tgcgcggcca
     1261 gccgggaacc gctcaactcg gccatgcgcg ggccgatctc ggcgaacacc gcccccgctt
     1321 cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc gtcgtccgcg acccacacct
     1381 tgccgatgtc gagcccgacg cgcgtgagga agagttcttg cagctcggtg acccgctcga
     1441 tgtggcggtc cggatcgacg gtgtggcgcg tggcggggta gtcggcgaac gcggcggcga
     1501 gggtgcgtac ggccctgggg acgtcgtcgc gggtggcgag gcgcaccgtg ggcttgtact
     1561 cggtcatggt acccacctga gggagggaaa atagaccaat aggcagagag agtcagtgcc
     1621 tatcagaaac ccaagagtct tctctgtctc cacgtgccca gtttctattg gtctccttaa
     1681 acctgtcttg taaccgaagt tcctatacta tctagagaat aggaacttct ctagataact
     1741 tcgtataatg tgtactatac gaagttatcg atggctgtcg aaaggcccgg agatgaggaa
     1801 gaggagaaca gcgcggcaga cgtgcgcttt tgaagcgtgc agaatgccgg gcctccggag
     1861 gaccttcggg cgcccgcccc gcccctgagc ccgcccctga gcccgccccc ggacccaccc
     1921 cttcccagcc tctgagccca gaaagcgaag gagcaaagct gctattggcc gctgccccaa
     1981 aggcctaccc gcttccattg ctcagcggtg ctgtccatct gcacgagact agtgagacgt
     2041 gctacttcca tttgtcacgt cctgcacgac gcgagctgcg gggcgggggg gaacttcctg
     2101 actaggggag gagtagaagg tggcgcgaag gggccaccaa agaacggagc cggttggcgc
     2161 ctaccggtgg atgtggaatg tgtgcgaggc cagaggccac ttgtgtagcg ccaagtgccc
     2221 agcggggctg ctaaagcgca tgctccagac tgccttggga aaagcgcctc ccctacccgg
     2281 gtgagataac ttcgtataag gtatcctata cgaagttatg cggccgcggt tacaagacag
     2341 gtttaaggag accaatagaa actgggcatg tggagacaga gaagactctt gggtttctga
     2401 taggcactga ctctctctgc ctattggtct attttcccac ccttagtgaa gagcgtttaa
     2461 acgcgatcgc gctcttcata agacctgcag gcatgcaagc ttggcactgg ccgtcgtttt
     2521 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2581 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2641 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2701 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     2761 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2821 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2881 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2941 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3001 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3061 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3121 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3181 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3241 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3301 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3361 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3421 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3481 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3541 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3601 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3661 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3721 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3781 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3841 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3901 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3961 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4021 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4081 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4141 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4201 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4261 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4321 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4381 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4441 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4501 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4561 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4621 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4681 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4741 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4801 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4861 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4921 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4981 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5041 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5101 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5161 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5221 cggttcctgg ccttttgctg gccttttgct ca
//
