LOCUS       sequence-433896-        7064 bp    DNA     circular SYN 15-APR-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 7064)
  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 7064)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: p204-U6-Switch-ON
FEATURES             Location/Qualifiers
     source          1..7064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     235..254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(473..577)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     645..663
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        763..1142
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1143..1346
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1297..1317
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1347..1522
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    1585..1784
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1985..2400
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2330..2352
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2368..2384
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     2491..2511
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2662..2681
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3117..3616
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3152..3175)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3549..3568
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3630..4433
                     /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(3693..3712)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4303..4322
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4604..5029
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5132..5154)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5290..5310)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5295..5624
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5475..5610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5646..5665
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5746..5763)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5917..6505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5997..6016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6676..7064,1..403,404..472))
                     /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"
ORIGIN
        1 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
       61 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
      121 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
      181 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
      241 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
      301 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
      361 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
      421 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      481 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      541 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      601 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
      661 ggccctttcg tgttcaagaa ttagcttggc cattgcatac gttgtatcca tatcataata
      721 tgtacattta tattggctca tgtccaacat taccgccatg ttgacattga ttattgacta
      781 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      841 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      901 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      961 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     1021 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1081 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1141 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     1201 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     1261 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     1321 ggtgggaggt ctatataagc agagctcaat aaaagagccc acaacccctc actcggcgcg
     1381 ccagtcctcc gatagactgc gtcgcccggg tacccgtatt cccaataaag cctcttgctg
     1441 tttgcatccg aatcgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
     1501 cccacgacgg gggtctttca tttgggggct cgtccgggat ttggagaccc ctgcccaggg
     1561 accaccgacc caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg
     1621 tctagtgtct atgtttgatg ttatgcgcct gcgtctgtac tagttagcta actagctctg
     1681 tatctggcgg acccgtggtg gaactgacga gttctgaaca cccggccgca accctgggag
     1741 acgtcccagg gactttgggg gccgtttttg tggcccgacc tgaggaaggg agtcgatgtg
     1801 gaatccgacc ccgtcaggat atgtggttct ggtaggagac gagaacctaa aacagttccc
     1861 gcctccgtct gaatttttgc tttcggtttg gaaccgaagc cgcgcgtctt gtctgctgca
     1921 gcgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt tgtctgaaaa
     1981 ttagggccag actgttacca ctcccttaag tttgacctta ggtcactgga aagatgtcga
     2041 gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttggttac cttctgctct
     2101 gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc
     2161 atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc
     2221 ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt
     2281 gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct
     2341 cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc
     2401 ggaattgaag atctccgcgg ttaattaaaa tgatacggcg accaccgaga tctgtcttga
     2461 tctaggagct actcatcaac ggtcacatgt gagggcctat ttcccatgat tccttcatat
     2521 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     2581 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2641 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatataact
     2701 tcgtatactt tatattatac gaagttattt tttttttttg cggccgccca gaattctggc
     2761 ctgcaggata acttcgtata ctttatatta tacgaagtta tgacgaaaca ccgggtcttc
     2821 gctcgagaga agacctgttt aagagctatg ctggaaacag catagcaagt ttaaataagg
     2881 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta
     2941 gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca
     3001 aatggctcta gaggtacccg ttaagatcgg aagagcacac gtctgaactc cagtcacaaa
     3061 attatctcgt atgccgtctt atgcttgtta attaacaatt gacgcgtaat tctaccgggt
     3121 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
     3181 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
     3241 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     3301 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     3361 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     3421 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     3481 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     3541 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     3601 atctccgggc ctttcgacct gcagccaata tgggatcggc cattgaacaa gatggattgc
     3661 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3721 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3781 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3841 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3901 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3961 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4021 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4081 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4141 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     4201 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4261 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4321 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4381 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgaggggatc
     4441 cgtcgacctg cagccaagct tatcgatacc gtcgagattt aattaagata tcatcagtgg
     4501 tccaggctct agttttgact caacaatatc accagctgaa gcctatagag tacgagccat
     4561 agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     4621 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac
     4681 tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagggtcg accctagaga
     4741 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     4801 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     4861 agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac
     4921 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     4981 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt
     5041 ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg taagctggct
     5101 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     5161 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     5221 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtaga
     5281 tccggctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     5341 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5401 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5461 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5521 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     5581 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt actggcttaa
     5641 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     5701 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     5761 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5821 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5881 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5941 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6001 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6061 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6121 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6181 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6241 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6301 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     6361 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6421 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6481 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6541 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6601 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6661 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6721 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6781 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6841 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6901 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6961 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     7021 tggtatggct tcattcagct ccggttccca acgatcaagg cgag
//
