LOCUS       sequence-434023-        5428 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 5428)
  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 5428)
  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-SWITCHER2-TF1-dsRED
FEATURES             Location/Qualifiers
     source          1..5428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    11..80
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     enhancer        88..373
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          641..868
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     881..900
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        881..899
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(944..977)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1350..1484
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1374..1393)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1428..1447
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1609..1642)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1656..2333
                     /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(1836..1856)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2248..2271
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2504..2552
                     /note="synthetic polyadenylation signal"
     misc_feature    2566..2657
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     2606..2625
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     repeat_region   2691..2831
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2691..2820
                     /label=AAV2 ITR
     rep_origin      2906..3361
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2993..3012)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3203..3224
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3378..3397)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3497..3519
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3557..3575)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3643..3747
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3748..3816,3817..4608)
                     /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(3966..3985)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4779..5367
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5268..5287
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aggtcacggt ctcgaagccg cggtgcgggt gccagggcgt gcccttgggc tccccgggcg
       61 cgtactccac ctcacccatc tggtacccgt tacataactt acggtaaatg gcccgcctgg
      121 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
      181 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      241 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
      301 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      361 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      421 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      481 gggggggggg ggggggggcg ccccgggggg ggggggcggg ggcggggcgg gggggggggg
      541 tgcgggggcg gccaatcagg gcggcgcccc ccgaaagttg ccttttatgg cgaggcggcg
      601 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct
      661 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc
      721 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag
      781 caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga
      841 gcacctgcct gaaatcactt tttttcaggt tggaccggtg taatacgact cactataggg
      901 aagcttccgt atacaaccta ctacctcata acatatgata tcgataactt cgtatagcat
      961 acattatacg aagttatccg cggccccgaa ttctttctct ggcctaactg gccggtacct
     1021 cgagaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg ccctggaagg
     1081 gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg gtcagagata
     1141 cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg
     1201 agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa
     1261 ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga
     1321 ttaagagctc gaattaattc atcgatgatg atccagacat gataagatac attgatgagt
     1381 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1441 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     1501 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaatga ggtagtaggt
     1561 tgtatacgga gcttccagtt tagccgtcga gccagtttag ccatcgacat aacttcgtat
     1621 agcatacatt atacgaagtt atactagtga tatccatggc ctcctccgag aacgtcatca
     1681 ccgagttcat gcgcttcaag gtgcgcatgg agggcaccgt gaacggccac gagttcgaga
     1741 tcgagggcga gggcgagggc cgcccctacg agggccacaa caccgtgaag ctgaaggtga
     1801 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct
     1861 ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg tccttccccg
     1921 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggcg accgtgaccc
     1981 aggactcctc cctgcaggac ggctgcttca tctacaaggt gaagttcatc ggcgtgaact
     2041 tcccctccga cggccccgtg atgcagaaga agaccatggg ctgggaggcc tccaccgagc
     2101 gcctgtaccc ccgcgacggc gtgctgaagg gcgagaccca caaggccctg aagctgaagg
     2161 acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag cccgtgcagc
     2221 tgcccggcta ctactacgtg gacgccaagc tggacatcac ctcccacaac gaggactaca
     2281 ccatcgtgga gcagtacgag cgcaccgagg gccgccacca cctgttcctg tagcgggata
     2341 tccgggattc gtcagtaggg ttgtaaaggt ttttcttttc ctgagaaaac aaccttttgt
     2401 tttctcaggt tttgcttttt ggcctttccc tagctttaaa aaaaaaggac gaaacaccac
     2461 gcgtctggcc taactggccg gtacctggta ttggacaggc cgcaataaaa tatctttatt
     2521 ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca
     2581 tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg
     2641 tgccagaaca tttctctgga tcctgccatg tgtccactga gcgcggccgc aggaacccct
     2701 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     2761 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     2821 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2881 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2941 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     3001 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3061 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3121 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3181 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     3241 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     3301 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     3361 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     3421 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3481 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3541 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3601 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3661 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3721 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3781 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3841 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3901 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3961 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4021 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4081 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4141 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4201 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4261 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4321 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4381 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4441 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     4501 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4561 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4621 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4681 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4741 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4801 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4861 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4921 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4981 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5041 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5101 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5161 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5221 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5281 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5341 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5401 gccttttgct ggccttttgc tcacatgt
//
