LOCUS       sequence-433897-        7099 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 7099)
  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 7099)
  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: p205-5'-Switch-ON
FEATURES             Location/Qualifiers
     source          1..7099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             419..844
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(947..969)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1105..1125)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1110..1439
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1290..1425
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1461..1480
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1561..1578)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1732..2320)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1812..1831)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2491..3282,3283..3351))
                     /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     3114..3133
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3352..3456)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3524..3542
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        3642..4021
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4022..4225
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4176..4196
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4226..4401
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    4464..4663
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             4864..5279
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     5209..5231
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     5247..5263
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        5370..5618
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5370..5390
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5541..5560
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(5620..5653)
                     /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(5693..5726)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6031..6530
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6066..6089)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6463..6482
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(6544..7099,1..248)
                     /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(6607..6626)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
       61 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
      121 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
      181 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
      241 tcttctgagg ggatccgtcg acctgcagcc aagcttatcg ataccgtcga gatttaatta
      301 agatatcatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta
      361 tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa
      421 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
      481 ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag
      541 ggtcgaccct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
      601 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
      661 cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact
      721 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
      781 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
      841 catttggggg ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg
      901 ggaggtaagc tggctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
      961 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     1021 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     1081 atagcggagt gtagatccgg ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     1141 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     1201 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     1261 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     1321 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     1381 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     1441 agcttactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     1501 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     1561 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1621 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     1681 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     1741 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     1801 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     1861 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     1921 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     1981 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2041 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2101 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2161 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2221 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2281 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2341 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2401 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2461 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2521 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2581 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2641 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2701 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2761 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt
     2821 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2881 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2941 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3001 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3061 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac
     3121 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3181 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3241 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3301 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3361 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3421 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3481 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     3541 gccctttcgt gttcaagaat tagcttggcc attgcatacg ttgtatccat atcataatat
     3601 gtacatttat attggctcat gtccaacatt accgccatgt tgacattgat tattgactag
     3661 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     3721 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     3781 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     3841 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     3901 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     3961 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     4021 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     4081 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     4141 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     4201 gtgggaggtc tatataagca gagctcaata aaagagccca caacccctca ctcggcgcgc
     4261 cagtcctccg atagactgcg tcgcccgggt acccgtattc ccaataaagc ctcttgctgt
     4321 ttgcatccga atcgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     4381 ccacgacggg ggtctttcat ttgggggctc gtccgggatt tggagacccc tgcccaggga
     4441 ccaccgaccc accaccggga ggtaagctgg ccagcaactt atctgtgtct gtccgattgt
     4501 ctagtgtcta tgtttgatgt tatgcgcctg cgtctgtact agttagctaa ctagctctgt
     4561 atctggcgga cccgtggtgg aactgacgag ttctgaacac ccggccgcaa ccctgggaga
     4621 cgtcccaggg actttggggg ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg
     4681 aatccgaccc cgtcaggata tgtggttctg gtaggagacg agaacctaaa acagttcccg
     4741 cctccgtctg aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag
     4801 cgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat
     4861 tagggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag
     4921 cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttggttacc ttctgctctg
     4981 cagaatggcc aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca
     5041 tcacccaggt taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc
     5101 cctacatcgt gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg
     5161 tacaccctaa gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc
     5221 ctcgttcgac cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccg
     5281 gaattgaaga tctccgcggt taattaaaat gatacggcga ccaccgagat ctgtcttgat
     5341 ctaggagcta ctcatcaacg gtcacatgtg agggcctatt tcccatgatt ccttcatatt
     5401 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     5461 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     5521 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     5581 tcttggcttt atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat
     5641 tatacgaagt tatttttttt ttttgcggcc gcccagaatt ctggcctgca ggataacttc
     5701 gtatagcata cattatacga agttatgggt cttcgctcga gagaagacct gtttaagagc
     5761 tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
     5821 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
     5881 tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta cccgttaaga
     5941 tcggaagagc acacgtctga actccagtca caaaattatc tcgtatgccg tcttatgctt
     6001 gttaattaac aattgacgcg taattctacc gggtagggga ggcgcttttc ccaaggcagt
     6061 ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc
     6121 ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct
     6181 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt
     6241 cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca
     6301 ccgctgagca atggaagcgg gtaggccttt ggggcggcgg ccaatagcag ctttgctcct
     6361 tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc
     6421 tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa
     6481 agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcagcc
     6541 aatatgggat cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     6601 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     6661 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     6721 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     6781 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     6841 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     6901 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     6961 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     7021 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     7081 cgcatgcccg acggcgagg
//
