LOCUS       sequence-287367-        6982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR - FLIP targeting vector for mouse beta-catenin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6982)
  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 6982)
  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-mCtnnb1-FLIP-Puro
FEATURES             Location/Qualifiers
     source          1..6982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1076..1111
                     /codon_start=1
                     /product="nanobody epitope tag corresponding to amino acids
                     16-27 of human beta-catenin (Traenkle et al., 2015)"
                     /label=BC2 tag
                     /translation="PDRKAAVSHWQQ"
     protein_bind    complement(1928..1961)
                     /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(2008..2041)
                     /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(2008..2041)
                     /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(2067..2086)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2121..2140
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2410..3009)
                     /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(2493..2513)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2990..3009
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    3143..3176
                     /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(3187..3686)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3235..3254)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3628..3651
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    3786..3819
                     /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    3786..3819
                     /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(4881..4898)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4881..4897)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4890..4912)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5110..5565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5197..5216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5407..5428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5582..5601)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5701..5723
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5761..5779)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5847..5951
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5952..6020,6021..6812)
                     /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(6170..6189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg
     1021 gggatcctct agagtcgtga cagctgacct gatggagttg gacatggcca tggagccgga
     1081 cagaaaagct gctgtcagcc actggcagca gcagtcttac ttggattctg gaatccattc
     1141 tggtgccacc accacagctc cttccctgag tggcaagggc aaccctgagg aagaagatgt
     1201 tgacacctcc caagtccttt atgaatggga gcaaggcttt tcccagtcct tcacgcaaga
     1261 gcaagtagct ggtaaagcat ttgtgtttgg aagctagcat taagtttctt gacagggctg
     1321 tgtgacagct cagccacagc acaagtgggt tgaaggaagg gcggagggta gcggagtccc
     1381 acatgcctag tgagtgttgg atttaccttt ttcagatatt gacgggcagt atgcaatgac
     1441 tagggctcag agggtccgag ctgccatgtt ccctgagacg ctagatgagg gcatgcagat
     1501 cccatccacg cagtttgacg ctgctcatcc cactaatgtc cagcgcttgg ctgaaccatc
     1561 acagatgttg aaacatgcag ttgtcaattt gattaactat caggatgacg cggaacttgc
     1621 cacacgtgca attcctgagc tgacaaaact gctaaacgat gaggaccagg taagcagtga
     1681 tgtaacttgc ttttcagttt actttgaggg aagttctcaa ggggatgcca atggctgcct
     1741 cacactaagt tttggaaact aatggtgcct cttcatcctt tattctaggt ggtagttaat
     1801 aaagctgctg ttatggtcca tcagctttcc aaaaaggaag cttccagaca tgccatcatg
     1861 cgctcccctc agatggtgtc tgccattgta cgcaccatgc aggtaagtat caaagatctg
     1921 gcgcgccata acttcgtata gtacacatta tacgaagtta tggctaagct ttgtgccatg
     1981 ccattgatgc tagcacagcc tgaatggata acttcgtata ggatacctta tacgaagtta
     2041 tgtcgacaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     2101 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     2161 gctgcaatta acaagtgcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     2221 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     2281 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     2341 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     2401 atcaggaatt caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac
     2461 ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc
     2521 ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac
     2581 gacggcgctg cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa
     2641 ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc
     2701 cagccgggaa ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc
     2761 ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac
     2821 cttgccgatg tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc
     2881 gatgtggcgg tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc
     2941 gagggtgcgt acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta
     3001 ctcggtcatg gtacccacct gagggaggga aaatagacca ataggcagag agagtcagtg
     3061 cctatcagaa acccaagagt cttctctgtc tccacgtgcc cagtttctat tggtctcctt
     3121 aaacctgtct tgtaacctct agataacttc gtataatgtg tactatacga agttatcgat
     3181 ggctgtcgaa aggcccggag atgaggaaga ggagaacagc gcggcagacg tgcgcttttg
     3241 aagcgtgcag aatgccgggc ctccggagga ccttcgggcg cccgccccgc ccctgagccc
     3301 gcccctgagc ccgcccccgg acccacccct tcccagcctc tgagcccaga aagcgaagga
     3361 gcaaagctgc tattggccgc tgccccaaag gcctacccgc ttccattgct cagcggtgct
     3421 gtccatctgc acgagactag tgagacgtgc tacttccatt tgtcacgtcc tgcacgacgc
     3481 gagctgcggg gcggggggga acttcctgac taggggagga gtagaaggtg gcgcgaaggg
     3541 gccaccaaag aacggagccg gttggcgcct accggtggat gtggaatgtg tgcgaggcca
     3601 gaggccactt gtgtagcgcc aagtgcccag cggggctgct aaagcgcatg ctccagactg
     3661 ccttgggaaa agcgcctccc ctacccgggt gaggcggccg cggttacaag acaggtttaa
     3721 ggagaccaat agaaactggg catgtggaga cagagaagac tcttgggttt ctgataggca
     3781 ctgacataac ttcgtataag gtatcctata cgaagttatt ttccctccct tagaatacaa
     3841 atgatgtaga gacagctcgt tgtactgctg ggactctgca caacctttct caccaccgcg
     3901 agggcttgct ggccatcttt aagtctggtg gcatcccagc gctggtgaaa atgcttgggt
     3961 aagagaatgg ccagggaact tgaagggtag gaagtagaag ggtgctggca tggctaacaa
     4021 aggctctcct cttcccaggt caccagtgga ttctgtactg ttctacgcca tcacgacact
     4081 gcataatctc ctgctccatc aggaaggagc taaaatggca gtgcgcctag ctggtggact
     4141 gcagaaaatg gttgctttgc tcaacaaaac aaacgtgaaa ttcttggcta ttacaacaga
     4201 ctgccttcag atcttagctt atggcaatca agagagcaag gtagagtgat gaaagttgtt
     4261 tgtacagagt gtggagttac taagttctta gtatgcatgt gcgcgcgcgc gcgcacacac
     4321 acacaggctc aaagatttgt gtctttagac actgaatctg tgtgtacagt acggtgctaa
     4381 gcattgaagg gatggggagc aggcctcata agtttacact tgggcagaga ggaggggctg
     4441 gatagacaga ggacatggtg ttttgaggtc ttgtggcaat ctttgagaag aagtgaggtg
     4501 ttacctagga ggaggcagtg agaggatgcc gtccacagtc ccgaggagga agagggatgg
     4561 tgtgctggag acacagtgtt agagttgggc taaggctggg ccgggtggat cctgtgaggc
     4621 tttctttgtt ggccatacca aagtccagga gataagatct taaacacagg catggggtga
     4681 gacctgccta tggaaatgtt caaggacctt aagggtgagg gagtggatat tctgcagtgt
     4741 ctcctaaggc aagtaacata gaagctcaga gtcagaagct aggggctctt agtgcccatt
     4801 ttcaactggc tgtttcgaca gaactttgat cacttctaca agcaaccctg ctttaagacc
     4861 tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4921 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4981 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     5041 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     5101 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5161 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5221 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5281 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     5341 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5401 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     5461 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5521 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     5581 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5641 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5701 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5761 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5821 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5881 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5941 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     6001 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6061 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6121 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6181 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6241 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6301 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6361 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6421 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6481 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6541 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6601 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6661 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6721 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6781 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6841 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6901 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6961 tagaaaagat caaaggatct tc
//
