LOCUS       sequence-434049-        7055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral Switch-OFF vector for sgRNA expression; 
            U6-BbsIx2-SWITCH-OFF-scaffold; neoR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7055)
  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 7055)
  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: p213-SWITCH-OFF
FEATURES             Location/Qualifiers
     source          1..7055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    9..42
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(84..332)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(142..161)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(312..332)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(423..838)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(439..455)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(471..493)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    1039..1238
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             1301..1476
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     promoter        complement(1477..1680)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1506..1526)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1681..2060
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2160..2178)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2246..2350
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2351..2419,2420..3211)
                     /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     complement(2569..2588)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3382..3970
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3871..3890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4124..4141
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4222..4241)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(4263..4592)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4277..4412
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4577..4597
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     4733..4755
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             4858..5283
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     CDS             complement(5454..6257)
                     /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(5565..5584)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6175..6194
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6271..6770)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6319..6338)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     6712..6735
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    6890..6923
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ctatgctgat aacttcgtat aatgtatgct atacgaagtt atcagcatag ctcttaaaca
       61 ggtcttctct cgagcgaaga cccggtgttt cgtcctttcc acaagatata taaagccaag
      121 aaatcgaaat actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt
      181 aaaactgcaa actacccaag aaattattac tttctacgtc acgtattttg tactaatatc
      241 tttgtgttta cagtcaaatt aattccaatt atctctctaa cagccttgta tcgtatatgc
      301 aaatatgaag gaatcatggg aaataggccc tcacatgtga ccgttgatga gtagctccta
      361 gatcaagaca gatctcggtg gtcgccgtat cattttaatt aaccgcggag atcttcaatt
      421 ccggcgccta gagaaggagt gagggctgga taaagggagg atcgaggcgg ggtcgaacga
      481 ggaggttcaa gggggagaga cggggcggat ggaggaagag gaggcggagg cttagggtgt
      541 acaaagggct tgacccaggg aggggggtca aaagccaagg cttcccaggt cacgatgtag
      601 gggacctggt ctgggtgtcc atgcgggcca ggtgaaaaga ccttgatctt aacctgggtg
      661 atgaggtctc ggttaaaggt gccgtctcgc ggccatccga cgttaaaggt tggccattct
      721 gcagagcaga aggtaaccaa cgtctcttct tgacatctac cgactggttg tgagcgatcc
      781 gctcgacatc tttccagtga cctaaggtca aacttaaggg agtggtaaca gtctggccct
      841 aattttcaga caaatacaga aacacagtca gacagagaca acacagaacg atgctgcagc
      901 gctgcagcag acaagacgcg cggcttcggt tccaaaccga aagcaaaaat tcagacggag
      961 gcgggaactg ttttaggttc tcgtctccta ccagaaccac atatcctgac ggggtcggat
     1021 tccacatcga ctcccttcct caggtcgggc cacaaaaacg gcccccaaag tccctgggac
     1081 gtctcccagg gttgcggccg ggtgttcaga actcgtcagt tccaccacgg gtccgccaga
     1141 tacagagcta gttagctaac tagtacagac gcaggcgcat aacatcaaac atagacacta
     1201 gacaatcgga cagacacaga taagttgctg gccagcttac ctcccggtgg tgggtcggtg
     1261 gtccctgggc aggggtctcc aaatcccgga cgagccccca aatgaaagac ccccgtcgtg
     1321 ggtagtcaat cactcagagg agaccctccc aaggaacagc gagaccacga ttcggatgca
     1381 aacagcaaga ggctttattg ggaatacggg tacccgggcg acgcagtcta tcggaggact
     1441 ggcgcgccga gtgaggggtt gtgggctctt ttattgagct ctgcttatat agacctccca
     1501 ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa
     1561 gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg
     1621 aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac
     1681 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
     1741 catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
     1801 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
     1861 ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga
     1921 cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt
     1981 aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata
     2041 actagtcaat aatcaatgtc aacatggcgg taatgttgga catgagccaa tataaatgta
     2101 catattatga tatggataca acgtatgcaa tggccaagct aattcttgaa cacgaaaggg
     2161 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2221 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2281 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2341 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2401 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2461 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2521 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2581 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2641 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2701 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2761 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2821 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2881 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2941 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3001 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3061 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3121 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3181 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3241 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3301 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3361 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3421 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3481 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3541 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3601 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3661 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3721 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3781 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3841 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3901 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3961 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4021 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4081 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4141 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4201 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtaagc
     4261 tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
     4321 gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     4381 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     4441 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     4501 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     4561 cctggggact ttccacaccc taactgacac acattccaca gccggatcta cactccgcta
     4621 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4681 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4741 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagcc agcttacctc
     4801 ccggtggtgg gtcggtggtc cctgggcagg ggtctcccga tcccggacga gcccccaaat
     4861 gaaagacccc cgctgacggg tagtcaatca ctcagaggag accctcccaa ggaacagcga
     4921 gaccacaagt cggatgcaac tgcaagaggg tttattggat acacgggtac ccgggcgact
     4981 cagtcaatcg gaggactggc gccccgagtg aggggttgtg ggctctttta ttgagctcgg
     5041 ggagcagaag cgcgcgaaca gaagcgagaa gcgaactgat tggttagttc aaataaggca
     5101 cagggtcatt tcaggtcctt ggggcaccct ggaaacatct gatggttctc tagggtcgac
     5161 cctgttccat ctgttcctga ccttgatctg aacttctcta ttctcagtta tgtatttttc
     5221 catgccttgc aaaatggcgt tacttaagct agcttgccaa acctacaggt ggggtctttc
     5281 attcccccct ttttctggag actaaataaa atcttttatt ttatctatgg ctcgtactct
     5341 ataggcttca gctggtgata ttgttgagtc aaaactagag cctggaccac tgatgatatc
     5401 ttaattaaat ctcgacggta tcgataagct tggctgcagg tcgacggatc ccctcagaag
     5461 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     5521 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     5581 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     5641 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     5701 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     5761 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     5821 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     5881 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     5941 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     6001 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     6061 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     6121 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     6181 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     6241 tcaatggccg atcccatatt ggctgcaggt cgaaaggccc ggagatgagg aagaggagaa
     6301 cagcgcggca gacgtgcgct tttgaagcgt gcagaatgcc gggcctccgg aggaccttcg
     6361 ggcgcccgcc ccgcccctga gcccgcccct gagcccgccc ccggacccac cccttcccag
     6421 cctctgagcc cagaaagcga aggagcaaag ctgctattgg ccgctgcccc aaaggcctac
     6481 ccgcttccat tgctcagcgg tgctgtccat ctgcacgaga ctagtgagac gtgctacttc
     6541 catttgtcac gtcctgcacg acgcgagctg cggggcgggg gggaacttcc tgactagggg
     6601 aggagtagaa ggtggcgcga aggggccacc aaagaacgga gccggttggc gcctaccggt
     6661 ggatgtggaa tgtgtgcgag gccagaggcc acttgtgtag cgccaagtgc ccagcggggc
     6721 tgctaaagcg catgctccag actgccttgg gaaaagcgcc tcccctaccc ggtagaatta
     6781 cgcgtcaatt gttaattaac aagcataaga cggcatacga gataattttg tgactggagt
     6841 tcagacgtgt gctcttccga tcttaacggg tacctctaga gccatttgta taacttcgta
     6901 taatgtatgc tatacgaagt tatctgcaga attggcgcac gcgctaaaaa cggactagcc
     6961 ttattttaac ttgctatttc tagctctaaa acaaaaaagc accgactcgg tgccactttt
     7021 tcaagttgat aacggactag ccttatttaa acttg
//
