LOCUS       sequence-434028-        4802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SWITCHER ready LoxP-EGFP reporter plasmid for cloning of a 
            customized CRISPR-inducible Cre-construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4802)
  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 4802)
  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: pLoxP-EGFP-crRNA-entry
FEATURES             Location/Qualifiers
     source          1..4802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     170..189
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   351..491
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   362..491
                     /label=AAV2 ITR
     misc_feature    525..616
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(557..576)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    630..678
                     /note="synthetic polyadenylation signal"
     CDS             complement(join(849..1562,1563..1565,1566..1568))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(852..1562,1563..1565,1566..1568))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(894..915)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1243..1262
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1502..1523
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1565..1574
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1587..1620
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1644..1663)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1645..1663)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1676..1903
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(1904..2181)
                     /label=chicken beta-actin promoter
     enhancer        2183..2468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     protein_bind    2476..2545
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     rep_origin      complement(2617..3205)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2697..2716)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3376..4167,4168..4236))
                     /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     3999..4018
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4237..4341)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4409..4427
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4465..4487)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4587..4606
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(4623..4802,1..276))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4760..4781)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
       61 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      121 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      181 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      241 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
      301 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
      361 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      421 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      481 taggggttcc tgcggccgcg ctcagtggac acatggcagg atccagagaa atgttctggc
      541 acctgcactt gcactgggga cagcctattt tgctagtttg ttttgtttcg ttttgttttg
      601 atggagagcg tatgttagta ctatcgattc acacaaaaaa ccaacacaca gatgtaatga
      661 aaataaagat attttattgc ggcctgtcca ataccaggta ccggccagtt aggccagacg
      721 cgtggtgttt cgtccttttt ttttaaagct agggaaaggc caaaaagcaa aacctgagaa
      781 aacaaaaggt tgttttctca ggaaaagaaa aacctttaca accctactga cgaatcccgg
      841 atatcgcttt acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
      901 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg
      961 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     1021 tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc
     1081 ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac
     1141 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     1201 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     1261 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     1321 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     1381 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     1441 agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt
     1501 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     1561 ctcaccatgg tggcgatatc actagtataa cttcgtataa tgtatgctat acgaagttat
     1621 gtcgacggct aaactggaag cttccctata gtgagtcgta ttacaccggt ccaacctgaa
     1681 aaaaagtgat ttcaggcagg tgctccaggt aattaaacat taatacccca ccaaccaacc
     1741 atcccttaaa cccttacctc ttgctcagct aattacagcc cggaggagaa gggccgtccc
     1801 gcccgctcac ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg
     1861 cggcggagcg gggcacgggg cgaaggcagc gcgcagcgac tcccgcccgc cgcgcgcttc
     1921 gctttttata gggccgccgc cgccgccgcc tcgccataaa aggaaacttt cggagcgcgc
     1981 cgctctgatt ggctgccgcc gcacctctcc gcctcgcccc gccccgcccc tcgccccgcc
     2041 ccgccccgcc tggcgcgcgc cccccccccc cccccgcccc catcgctgca caaaataatt
     2101 aaaaaataaa taaatacaaa attgggggtg gggagggggg ggagatgggg agagtgaagc
     2161 agaacgtggg gctcacctcg accatggtaa tagcgatgac taatacgtag atgtactgcc
     2221 aagtaggaaa gtcccataag gtcatgtact gggcacaatg ccaggcgggc catttaccgt
     2281 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     2341 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     2401 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     2461 atgtaacggg taccagatgg gtgaggtgga gtacgcgccc ggggagccca agggcacgcc
     2521 ctggcacccg caccgcggct tcgagaccgt gacctacatg tgagcaaaag gccagcaaaa
     2581 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2641 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2701 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2761 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2821 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2881 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2941 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3001 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3061 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3121 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3181 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3241 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3301 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3361 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3421 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3481 cttaccatct ggccccagtg ctgcaatgat accgcggctt ccacgctcac cggctccaga
     3541 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3601 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3661 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3721 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3781 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3841 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3901 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3961 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4021 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4081 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4141 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4201 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4261 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4321 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4381 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     4441 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4501 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4561 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4621 taaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     4681 attttttaac caatagaccg aaatcggcaa aatcccttat aaatcaaaag aatagcccga
     4741 gatagagttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4801 ca
//
