LOCUS       sequence-433898-        7093 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 7093)
  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 7093)
  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: p206-Switch-ON
FEATURES             Location/Qualifiers
     source          1..7093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    4..203
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             404..819
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     749..771
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     787..803
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        910..1158
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     910..930
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1081..1100
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1200..1233)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1273..1306)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1566..2065
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1601..1624)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1998..2017
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2079..2882
                     /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(2142..2161)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2752..2771
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             3053..3477
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(3580..3602)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3738..3758)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3743..4072
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3923..4058
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4094..4113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4194..4211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4365..4953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4445..4464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5124..5915,5916..5984))
                     /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     5747..5766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5985..6089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6157..6175
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        6275..6654
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6655..6858
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6809..6829
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6859..7034
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
ORIGIN
        1 ggtaagctgg ccagcaactt atctgtgtct gtccgattgt ctagtgtcta tgtttgatgt
       61 tatgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga cccgtggtgg
      121 aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg actttggggg
      181 ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc cgtcaggata
      241 tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg aatttttgct
      301 ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca tcgttctgtg
      361 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac
      421 tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc acaaccagtc
      481 ggtagatgtc aagaagagac gttggttacc ttctgctctg cagaatggcc aacctttaac
      541 gtcggatggc cgcgagacgg cacctttaac cgagacctca tcacccaggt taagatcaag
      601 gtcttttcac ctggcccgca tggacaccca gaccaggtcc cctacatcgt gacctgggaa
      661 gccttggctt ttgacccccc tccctgggtc aagccctttg tacaccctaa gcctccgcct
      721 cctcttcctc catccgcccc gtctctcccc cttgaacctc ctcgttcgac cccgcctcga
      781 tcctcccttt atccagccct cactccttct ctaggcgccg gaattgaaga tctccgcggt
      841 taattaaaat gatacggcga ccaccgagat ctgtcttgat ctaggagcta ctcatcaacg
      901 gtcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      961 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1021 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1081 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     1141 tggaaaggac gaaacaccgg gtcttcgctc gagagaagac ctgtttaaga gctatgctga
     1201 taacttcgta tagcatacat tatacgaagt tatttttttt ttttgcggcc gcccagaatt
     1261 ctggcctgca ggataacttc gtatagcata cattatacga agttatcagc atagcaagtt
     1321 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
     1381 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
     1441 ctgcagacaa atggctctag aggtacccgt taagatcgga agagcacacg tctgaactcc
     1501 agtcacaaaa ttatctcgta tgccgtctta tgcttgttaa ttaacaattg acgcgtaatt
     1561 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     1621 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     1681 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     1741 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     1801 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     1861 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     1921 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     1981 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     2041 ctcttcctca tctccgggcc tttcgacctg cagccaatat gggatcggcc attgaacaag
     2101 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2161 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     2221 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     2281 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     2341 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     2401 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     2461 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     2521 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     2581 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     2641 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     2701 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     2761 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     2821 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     2881 gaggggatcc gtcgacctgc agccaagctt atcgataccg tcgagattta attaagatat
     2941 catcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag cctatagagt
     3001 acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     3061 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
     3121 atacataact gagaatagag aagttcagat caaggtcagg aacagatgga acagggtcga
     3181 ccctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     3241 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     3301 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg
     3361 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     3421 gttccttggg agggtctcct ctgagtgatt gactacccac gacgggggtc tttcatttgg
     3481 gggctcgtcc gggatttgga gacccctgcc cagggaccac cgacccacca ccgggaggta
     3541 agctggctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3601 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3661 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     3721 agtgtagatc cggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3781 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3841 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3901 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3961 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4021 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagcttac
     4081 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     4141 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4801 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5401 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     5461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     5761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6121 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     6181 cgtgttcaag aattagcttg gccattgcat acgttgtatc catatcataa tatgtacatt
     6241 tatattggct catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa
     6301 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     6361 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     6421 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     6481 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     6541 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     6601 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     6661 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     6721 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     6781 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     6841 gtctatataa gcagagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct
     6901 ccgatagact gcgtcgcccg ggtacccgta ttcccaataa agcctcttgc tgtttgcatc
     6961 cgaatcgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccacgac
     7021 gggggtcttt catttggggg ctcgtccggg atttggagac ccctgcccag ggaccaccga
     7081 cccaccaccg gga
//
