LOCUS       sequence-100354-        5999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinase-mediated cassette exchange in ES cells to insert the Dre
            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 5999)
  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 5999)
  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-Dre Flp-in replacement vector
FEATURES             Location/Qualifiers
     source          1..5999
                     /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             807..827
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             828..1850
                     /product="site-specific recombinase"
                     /label=Dre recombinase
                     /note="Dre is a site-specific recombinase from 
                     bacteriophage D6. Recombination occurs at rox sequences."
     protein_bind    2571..2604
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        2631..3130
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2666..2689)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3063..3082
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(3778..3794)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3778..3794)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3791..3813)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3802..3818
                     /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(3826..3832,3833..3850,3851..3856))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3871..3892
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4009..4026)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4180..4768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4260..4279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4939..5730,5731..5799))
                     /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     5562..5581
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5800..5904)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5972..5990
                     /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 cctatgccta agaagaagag gaaggtttct gagctgatta
      841 ttagtggttc atctggtgga ttcctgcgaa acatcggcaa agagtatcag gaggccgctg
      901 aaaacttcat gaggtttatg aatgaccagg gggcgtacgc tcctaacact ttgagggatt
      961 tgaggttggt ctttcatagc tgggccagat ggtgccatgc tcggcagctt gcatggtttc
     1021 caattagtcc tgaaatggca cgcgaatact ttcttcagtt gcacgatgca gacctggcct
     1081 ccactaccat cgacaagcac tatgctatgc ttaatatgct tctgtcccac tgcggactgc
     1141 cacccttgtc cgacgacaag tcagtgagtc ttgccatgag aagaattaga agagaagccg
     1201 caaccgaaaa gggtgagagg acaggacagg caatccccct gcgctgggac gacctgaagc
     1261 tgctggatgt gctgctcagc aggagcgagc ggctggtcga cctgcgcaac agggctttcc
     1321 tgttcgtagc ctataacacc ctcatgagaa tgtctgaaat atcacgcatc agggttgggg
     1381 acttggatca gacaggagac acagtgaccc tgcacatcag tcacactaag acaatcacca
     1441 cagctgcggg ccttgacaaa gtgctctccc ggcgaaccac agcagtgctc aatgactggc
     1501 tggacgtcag tgggcttaga gaacatccag acgctgtgct cttcccacct atacaccggt
     1561 caaacaaagc ccgcattact accacgcccc tgaccgcccc tgccatggag aagattttca
     1621 gtgatgcctg ggtgctgctg aacaaacggg acgccacccc caataaaggg aggtatagga
     1681 cctggaccgg ccattccgcc agggtgggtg ccgcaataga catggccgag aaacaggtgt
     1741 ctatggtcga gattatgcag gaagggacat ggaagaagcc tgaaacactg atgcggtatc
     1801 tcagaagggg cggagtgtcc gtgggagcca attctcgact gatggatagc taagtggcgc
     1861 tgactgcttg ggtcccccac ctctccatcc ccaacgcccc atctcagtcc ttctcgcggc
     1921 cctcctgagt ttctgttcag tttgtttgtg ttatttttta ctcccccatc ctctatggcc
     1981 ctcggatgac gccattcttc tggaaatgct ggagaaacaa taaaggctgt accaatcgga
     2041 caccacctgt agggaggacc caggcctggc agggtgttgg tttggcaagt tttttttctt
     2101 tctttttagg gcagtggggg tatagtagaa aaagtgagat aagtcaaagg acaacgcccc
     2161 gatatctcct gcctgcttgg tactgagtgc tcatgtgggc cacctcgttc aatctctgca
     2221 cctttcccac aaggagatgg gggtgatgga tcgtccatct taaagataca gaaactgcct
     2281 tttaaagagc agaagggaag ggaaggggtt gagtccttca ggactagcta gatcaaagga
     2341 ctccaatgac actctatcaa ttgcttttga ctttgctgtg ataaaatacc tgaaaagaga
     2401 aacttaagga aagagcggtt tcttttggct ctctaagagg gtgcggccta tcattggtag
     2461 ggaagcccca ggaggcaggc tacattgtgt cacactgtat ccacattcag gaaggaaggc
     2521 gcgccgtaaa agcttggcat agatcttatt gcagaagcca tatgttttag gtgccagggc
     2581 gtgcccttgg gctccccggg cgcgtttggg cggccgcatc gaattctacc gggtagggga
     2641 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
     2701 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc
     2761 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
     2821 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
     2881 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
     2941 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
     3001 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
     3061 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
     3121 gggcctttcg acctgcaatc gccgctagcc gccaccatga aaaagcctga actcaccgcg
     3181 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     3241 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     3301 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     3361 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     3421 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     3481 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     3541 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     3601 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     3661 accgtcagtg taagtgctag cccctaggcc tgaatcgaag ttcctattct tcaaaaggta
     3721 taggaacttc gagctcgtcg actgcagagg cctgcatgca agcttggcgt aatcatggtc
     3781 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3841 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3901 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3961 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4021 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4081 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4141 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4201 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4261 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4321 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4381 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4441 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4501 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4561 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4621 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4681 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4741 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4801 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4861 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4921 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4981 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5041 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5101 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5161 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5221 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5281 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5341 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5401 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5461 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5521 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5581 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5641 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5701 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5761 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5821 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5881 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     5941 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
