LOCUS       sequence-434016-        6208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CRISPR/Cas12a-inducible Split-Cre construct with dsRED as a 
            reporter after the Lox-Stop-Lox cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6208)
  AUTHORS   Schuster B, Dobiasovska I, Curcic J, Pajer P, Borna S, Bartunek P
  TITLE     SWITCHER, a CRISPR-inducible floxed wild-type Cre regulating CRISPR 
            activity.
  JOURNAL   Commun Biol. 2025 Jul 2;8(1):982. doi: 10.1038/s42003-025-08427-8.
  PUBMED    40596718
REFERENCE   2  (bases 1 to 6208)
  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: "Commun 
            Biol."; date: "2025-07-2"; volume: "8(1):982. doi"; pages: " 
            10.1038/s42003-025-08427-8"
COMMENT     Sequence Label: pSplit-SWITCHER1-TF1-dsRED
FEATURES             Location/Qualifiers
     source          1..6208
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(15..34)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     69..88
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(250..283)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             297..974
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(477..497)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     889..912
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1145..1193
                     /note="synthetic polyadenylation signal"
     misc_feature    1207..1298
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1247..1266
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     repeat_region   1332..1472
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1332..1461
                     /label=AAV2 ITR
     rep_origin      1547..2002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1634..1653)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1844..1865
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2019..2038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2138..2160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2198..2216)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2284..2388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2389..2457,2458..3249)
                     /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(2607..2626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3420..4008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3909..3928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4080..4149
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     enhancer        4157..4442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          4844..5071
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     5084..5103
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5084..5102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(5147..5180)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    join(6199..6208,1..125)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
       61 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
      121 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
      181 ttttttaatg aggtagtagg ttgtatacgg agcttccagt ttagccgtcg agccagttta
      241 gccatcgaca taacttcgta tagcatacat tatacgaagt tatactagtg atatccatgg
      301 cctcctccga gaacgtcatc accgagttca tgcgcttcaa ggtgcgcatg gagggcaccg
      361 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggccaca
      421 acaccgtgaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
      481 ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact
      541 acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
      601 gcggcgtggc gaccgtgacc caggactcct ccctgcagga cggctgcttc atctacaagg
      661 tgaagttcat cggcgtgaac ttcccctccg acggccccgt gatgcagaag aagaccatgg
      721 gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagaccc
      781 acaaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag tccatctaca
      841 tggccaagaa gcccgtgcag ctgcccggct actactacgt ggacgccaag ctggacatca
      901 cctcccacaa cgaggactac accatcgtgg agcagtacga gcgcaccgag ggccgccacc
      961 acctgttcct gtagcgggat atccgggatt cgtcagtagg gttgtaaagg tttttctttt
     1021 cctgagaaaa caaccttttg ttttctcagg ttttgctttt tggcctttcc ctagctttaa
     1081 aaaaaaagga cgaaacacca cgcgtctggc ctaactggcc ggtacctggt attggacagg
     1141 ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat
     1201 agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc
     1261 tgtccccagt gcaagtgcag gtgccagaac atttctctgg atcctgccat gtgtccactg
     1321 agcgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     1381 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     1441 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     1501 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     1561 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1621 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1681 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1741 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     1801 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1861 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     1921 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1981 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     2041 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2101 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2161 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     2221 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     2281 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2341 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2401 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2461 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2521 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2581 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2641 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2701 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2761 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2821 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2881 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2941 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3001 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3061 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     3121 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3181 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3241 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3301 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3361 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3421 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3481 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3541 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3601 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3661 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3721 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3781 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3841 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3901 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3961 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4021 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgta ggtcacggtc
     4081 tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc
     4141 tcacccatct ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     4201 acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat
     4261 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     4321 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca
     4381 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     4441 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     4501 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
     4561 gggggggggc ccgggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     4621 gggggggggg ggccgcccag gcggggcggg gcgggggggg gggggggtgg gggggggggg
     4681 gggggggcgg ggcccgggga gagcggaagg cggggggggg gggggcgggg gggggcgcgg
     4741 cggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     4801 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     4861 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     4921 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     4981 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     5041 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgtaatacg actcactata
     5101 gggaagcttc cgtatacaac ctactacctc ataacatatg atatcgataa cttcgtatag
     5161 catacattat acgaagttat ccgcgggccc gggatccacc ggtcgccgcc accatgtcca
     5221 atttactgac cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg
     5281 ttcgcaagaa cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga
     5341 aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat
     5401 ggtttccccc agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc
     5461 tggcagtaaa aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg
     5521 ggctgccacg accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag
     5581 aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg
     5641 accaggttcg ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat
     5701 ttctggggat tgcttataac accctgttac gtatagccga aattgccagg atcagggtta
     5761 aagatatctc acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc
     5821 tggttagcac cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc
     5881 gatggatttc cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca
     5941 gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag
     6001 ggatttttga agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat
     6061 acctggcctg gtctggacac agtgcccgtg tcggagccgc gcgagattga attctttctc
     6121 tggcctaact ggccggtacc tcgagccgta tacaacctac tacctcattc ccagagctcg
     6181 aattaattca tcgatgatga tccagaca
//
