LOCUS       sequence-100355-        6248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinase-mediated cassette exchange in ES cells to insert the 
            Flpo recombinase gene at the mouse Pvalb gene stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6248)
  AUTHORS   Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, Li L, van
            der Bourg A, Niino Y, Egolf L, Monetti C, Gu H, Mills M, Cheng A, 
            Tasic B, Nguyen TN, Sunkin SM, Benucci A, Nagy A, Miyawaki A, 
            Helmchen F, Empson RM, Knopfel T, Boyden ES, Reid RC, Carandini M, 
            Zeng H
  TITLE     Transgenic mice for intersectional targeting of neural sensors and 
            effectors with high specificity and performance.
  JOURNAL   Neuron. 2015 Mar 4;85(5):942-58. doi: 10.1016/j.neuron.2015.02.022.
  PUBMED    25741722
REFERENCE   2  (bases 1 to 6248)
  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"; doi: 
            "10.1016/j.neuron.2015.02"; journalName: "Neuron"; date: "2015-03-4-
            4"; volume: "85"; issue: "5"; pages: "942-58"
COMMENT     Sequence Label: Pvalb-2A-Flpo Flp-in replacement vector
FEATURES             Location/Qualifiers
     source          1..6248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    414..447
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             750..803
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(804..809,810..830,831..2102)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     protein_bind    2820..2853
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        2880..3379
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2915..2938)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3312..3331
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(4027..4043)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4027..4043)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4040..4062)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4051..4067
                     /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)."
     promoter        complement(join(4075..4081,4082..4099,4100..4105))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4120..4141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4258..4275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4429..5017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4509..4528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5188..5979,5980..6048))
                     /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     5811..5830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6049..6153)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6221..6239
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accgaagttc
      421 ctattcttca aatagtatag gaacttcttt cggccggcct cagggggttc tcagagacaa
      481 tgaatgacga gttctaagtc taccctgacc cctccggtca aacagcagct gttcaaagaa
      541 acggccaggc tcagagcctc cattccctca tccacagagc aggcatggtg actaacaaca
      601 cagtcgcagc tgaagcccaa ggagatattg tactcggagg gtttaagata gctggtgcac
      661 ggcagacgtg gggatgcttg ccgaagataa gagtgtgtca tttttctttt gcagaattct
      721 ccactctggt ggctgaaagc ggttccggag agggcagagg aagtcttcta acatgcggtg
      781 acgtggagga gaatcccggc cctatggctc ctaagaagaa gaggaaggtg atgagccagt
      841 tcgacatcct gtgcaagacc ccccccaagg tgctggtgcg gcagttcgtg gagagattcg
      901 agaggcccag cggcgagaag atcgccagct gtgccgccga gctgacctac ctgtgctgga
      961 tgatcaccca caacggcacc gccatcaaga gggccacctt catgagctac aacaccatca
     1021 tcagcaacag cctgagcttc gacatcgtga acaagagcct gcagttcaag tacaagaccc
     1081 agaaggccac catcctggag gccagcctga agaagctgat ccccgcctgg gagttcacca
     1141 tcatccctta caacggccag aagcaccaga gcgacatcac cgacatcgtg tccagcctgc
     1201 agctgcagtt cgagagcagc gaggaggccg acaagggcaa cagccacagc aagaagatgc
     1261 tgaaggccct gctgtccgag ggcgagagca tctgggagat caccgagaag atcctgaaca
     1321 gcttcgagta caccagcagg ttcaccaaga ccaagaccct gtaccagttc ctgttcctgg
     1381 ccacattcat caactgcggc aggttcagcg acatcaagaa cgtggacccc aagagcttca
     1441 agctggtgca gaacaagtac ctgggcgtga tcattcagtg cctggtgacc gagaccaaga
     1501 caagcgtgtc caggcacatc tactttttca gcgccagagg caggatcgac cccctggtgt
     1561 acctggacga gttcctgagg aacagcgagc ccgtgctgaa gagagtgaac aggaccggca
     1621 acagcagcag caacaagcag gagtaccagc tgctgaagga caacctggtg cgcagctaca
     1681 acaaggccct gaagaagaac gccccctacc ccatcttcgc tatcaagaac ggccctaaga
     1741 gccacatcgg caggcacctg atgaccagct ttctgagcat gaagggcctg accgagctga
     1801 caaacgtggt gggcaactgg agcgacaaga gggcctccgc cgtggccagg accacctaca
     1861 cccaccagat caccgccatc cccgaccact acttcgccct ggtgtccagg tactacgcct
     1921 acgaccccat cagcaaggag atgatcgccc tgaaggacga gaccaacccc atcgaggagt
     1981 ggcagcacat cgagcagctg aagggcagcg ccgagggcag catcagatac cccgcctgga
     2041 acggcatcat cagccaggag gtgctggact acctgagcag ctacatcaac aggcggatct
     2101 gagtggcgct gactgcttgg gtcccccacc tctccatccc caacgcccca tctcagtcct
     2161 tctcgcggcc ctcctgagtt tctgttcagt ttgtttgtgt tattttttac tcccccatcc
     2221 tctatggccc tcggatgacg ccattcttct ggaaatgctg gagaaacaat aaaggctgta
     2281 ccaatcggac accacctgta gggaggaccc aggcctggca gggtgttggt ttggcaagtt
     2341 ttttttcttt ctttttaggg cagtgggggt atagtagaaa aagtgagata agtcaaagga
     2401 caacgccccg atatctcctg cctgcttggt actgagtgct catgtgggcc acctcgttca
     2461 atctctgcac ctttcccaca aggagatggg ggtgatggat cgtccatctt aaagatacag
     2521 aaactgcctt ttaaagagca gaagggaagg gaaggggttg agtccttcag gactagctag
     2581 atcaaaggac tccaatgaca ctctatcaat tgcttttgac tttgctgtga taaaatacct
     2641 gaaaagagaa acttaaggaa agagcggttt cttttggctc tctaagaggg tgcggcctat
     2701 cattggtagg gaagccccag gaggcaggct acattgtgtc acactgtatc cacattcagg
     2761 aaggaaggcg cgccgtaaaa gcttggcata gatcttattg cagaagccat atgttttagg
     2821 tgccagggcg tgcccttggg ctccccgggc gcgtttgggc ggccgcatcg aattctaccg
     2881 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     2941 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     3001 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     3061 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     3121 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     3181 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     3241 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     3301 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     3361 ctcatctccg ggcctttcga cctgcaatcg ccgctagccg ccaccatgaa aaagcctgaa
     3421 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     3481 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     3541 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     3601 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     3661 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     3721 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     3781 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     3841 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     3901 atggacgaca ccgtcagtgt aagtgctagc ccctaggcct gaatcgaagt tcctattctt
     3961 caaaaggtat aggaacttcg agctcgtcga ctgcagaggc ctgcatgcaa gcttggcgta
     4021 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4081 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4141 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4201 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4261 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4321 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4381 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4441 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4501 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4561 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4621 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4681 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4741 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4801 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4861 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4921 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4981 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5041 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5101 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5161 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5221 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5281 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5341 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5401 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5461 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5521 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5581 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5641 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5701 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5761 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5821 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5881 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5941 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6001 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6061 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6121 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6181 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6241 ctttcgtc
//
