LOCUS       sequence-434054-        7094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral Switch-ON vector for sgRNA expression; 
            U6-BbsIx2-SWITCH-ON-scaffold; neoR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7094)
  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 7094)
  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: p207-Switch-ON (FRT)
FEATURES             Location/Qualifiers
     source          1..7094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..47
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        307..806
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(342..365)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     739..758
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             820..1623
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(883..902)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1493..1512
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             1794..2219
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(2322..2344)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2480..2500)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2485..2814
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2665..2800
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2836..2855
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2936..2953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3107..3695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3187..3206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3866..4657,4658..4726))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4489..4508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4727..4831)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4899..4917
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        5017..5396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5397..5600
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5551..5571
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5601..5776
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    5839..6038
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6239..6654
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6584..6606
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6622..6638
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        6745..6993
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6745..6765
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6916..6935
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    7035..7068
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
ORIGIN
        1 tctggcctgc agggaagttc ctattctcta gaaagtatag gaacttccag catagcaagt
       61 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
      121 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      181 tctgcagaca aatggctcta gaggtacccg ttaagatcgg aagagcacac gtctgaactc
      241 cagtcacaaa attatctcgt atgccgtctt atgcttgtta attaacaatt gacgcgtaat
      301 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
      361 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
      421 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
      481 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
      541 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
      601 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
      661 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
      721 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
      781 cctcttcctc atctccgggc ctttcgacct gcagccaata tgggatcggc cattgaacaa
      841 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
      901 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
      961 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     1021 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     1081 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1141 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1201 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1261 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1321 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     1381 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     1441 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     1501 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     1561 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     1621 tgaggggatc cgtcgacctg cagccaagct tatcgatacc gtcgagattt aattaagata
     1681 tcatcagtgg tccaggctct agttttgact caacaatatc accagctgaa gcctatagag
     1741 tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa
     1801 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa
     1861 aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagggtcg
     1921 accctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
     1981 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
     2041 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
     2101 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
     2161 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
     2221 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
     2281 taagctggct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     2341 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     2401 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     2461 ggagtgtaga tccggctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2521 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2581 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2641 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2701 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     2761 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt
     2821 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     2881 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     2941 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3001 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3061 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3121 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3181 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3241 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3301 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3361 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3421 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3481 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3541 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3601 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3661 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3721 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3781 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3841 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3901 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3961 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4021 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4081 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4141 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     4201 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4261 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4321 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4381 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4441 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     4501 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4561 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4621 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4681 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4741 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4801 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4861 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4921 ttcgtgttca agaattagct tggccattgc atacgttgta tccatatcat aatatgtaca
     4981 tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt
     5041 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     5101 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     5161 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     5221 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     5281 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     5341 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     5401 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     5461 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     5521 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     5581 aggtctatat aagcagagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     5641 ctccgataga ctgcgtcgcc cgggtacccg tattcccaat aaagcctctt gctgtttgca
     5701 tccgaatcgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccacg
     5761 acgggggtct ttcatttggg ggctcgtccg ggatttggag acccctgccc agggaccacc
     5821 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
     5881 gtctatgttt gatgttatgc gcctgcgtct gtactagtta gctaactagc tctgtatctg
     5941 gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg ggagacgtcc
     6001 cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga tgtggaatcc
     6061 gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc
     6121 gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc tgcagcgctg
     6181 cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg aaaattaggg
     6241 ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat
     6301 cgctcacaac cagtcggtag atgtcaagaa gagacgttgg ttaccttctg ctctgcagaa
     6361 tggccaacct ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc
     6421 caggttaaga tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac
     6481 atcgtgacct gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac
     6541 cctaagcctc cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt
     6601 tcgaccccgc ctcgatcctc cctttatcca gccctcactc cttctctagg cgccggaatt
     6661 gaagatctcc gcggttaatt aaaatgatac ggcgaccacc gagatctgtc ttgatctagg
     6721 agctactcat caacggtcac atgtgagggc ctatttccca tgattccttc atatttgcat
     6781 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     6841 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     6901 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     6961 gctttatata tcttgtggaa aggacgaaac accgggtctt cgctcgagag aagacctgtt
     7021 taagagctat gctggaagtt cctattctct agaaagtata ggaacttctt tttttttttg
     7081 cggccgccca gaat
//
