LOCUS       sequence-434053-        3970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  insert vector for Switch-OVER: Single loxP-STOP-EF1a-Puro-mU6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3970)
  AUTHORS   Chylinski K, Hubmann M, Hanna RE, Yanchus C, Michlits G, Uijttewaal 
            ECH, Doench J, Schramek D, Elling U
  TITLE     CRISPR-Switch regulates sgRNA activity by Cre recombination for 
            sequential editing of two loci.
  JOURNAL   Nat Commun. 2019 Nov 29;10(1):5454. doi: 10.1038/s41467-019-13403-y.
  PUBMED    31784531
REFERENCE   2  (bases 1 to 3970)
  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: "Nat 
            Commun."; date: "2019-11-29"; volume: "10(1):5454. doi"; pages: " 
            10.1038/s41467-019-13403-y"
COMMENT     Sequence Label: p944-SWITCH-OVER insert: EF1a-Puro
FEATURES             Location/Qualifiers
     source          1..3970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..118
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(156..178)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     278..297
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     491..513
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     505..522
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     506..522
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(569..882)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(630..650)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     CDS             complement(883..1482)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(966..986)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1463..1482
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(1507..1718)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    1784..1817
                     /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(1876..1892)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1876..1892)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1889..1911)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1900..1916
                     /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(1924..1930,1931..1948,1949..1954))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1969..1990
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2107..2124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2278..2866)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2358..2377)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3037..3828,3829..3897))
                     /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     3660..3679
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(3898..3970,1..32))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
       61 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
      121 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
      181 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
      241 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
      301 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
      361 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
      421 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
      481 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattcga gctcggtacc
      541 cggggatcct ctagagtcga agaccccaaa caaggctttt ctccaaggga tatttatagt
      601 ctcaaaacac acaattactt tacagttagg gtgagtttcc ttttgtgctg ttttttaaaa
      661 taataattta gtatttgtat ctcttataga aatccaagcc tatcatgtaa aatgtagcta
      721 gtattaaaaa gaacagatta tctgtctttt atcgcacatt aagcctctat agttactagg
      781 aaatattata tgcaaattaa ccggggcagg ggagtagccg agcttctccc acaagtctgt
      841 gcgagggggc cggcgcgggc ctagagatgg cggcgtcgga tctcaggcac cgggcttgcg
      901 ggtcatgcac caggtgcgcg gtccttcggg cacctcgacg tcggcggtga cggtgaagcc
      961 gagccgctcg tagaagggga ggttgcgggg cgcggaggtc tccaggaagg cgggcacccc
     1021 ggcgcgctcg gccgcctcca ctccggggag cacgacggcg ctgcccagac ccttgccctg
     1081 gtggtcgggc gagactccga cggtggccag gaaccacgcg ggctccttgg gccggtgcgg
     1141 cgccaggagg ccttccatct gttgctgcgc ggccagccgg gaaccgctca actcggccat
     1201 gcgcgggccg atctcggcga acaccgcccc cgcttcgacg ctctccggcg tggtccagac
     1261 cgccaccgcg gcgccgtcgt ccgcgaccca caccttgccg atgtcgagcc cgacgcgcgt
     1321 gaggaagagt tcttgcagct cggtgacccg ctcgatgtgg cggtccggat cgacggtgtg
     1381 gcgcgtggcg gggtagtcgg cgaacgcggc ggcgagggtg cgtacggccc tggggacgtc
     1441 gtcgcgggtg gcgaggcgca ccgtgggctt gtactcggtc atggtggcag cgctctagaa
     1501 ccggtcctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc
     1561 acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact
     1621 ttcccagttt accccgcgcc accttctcta ggcaccggat caattgccga cccctccccc
     1681 caacttctcg gggactgtgg gcgatgtgcg ctctgcccac tgacgggcac cggagccaat
     1741 tcccactcca cgcgtcaatt ctgggcggcc gcaaaaaaaa aaaataactt cgtataatgt
     1801 atgctatacg aagttatcag catagctctt aaacaggtct tcgacctgca ggcatgcaag
     1861 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     1921 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     1981 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2041 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2101 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2161 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2221 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2281 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2341 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2401 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2461 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2521 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2581 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2641 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2701 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2761 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2821 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2881 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2941 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3001 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3061 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3121 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3181 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3241 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3301 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3361 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3421 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3481 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3541 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3601 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3661 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3721 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3781 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3841 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3901 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3961 atttgaatgt
//
