LOCUS       sequence-441235-        8159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-screening.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8159)
  AUTHORS   Uijttewaal ECH, Lee J, Sell AC, Botay N, Vainorius G, Novatchkova M,
            Baar J, Yang J, Potzler T, van der Leij S, Lowden C, Sinner J, 
            Elewaut A, Gavrilovic M, Obenauf A, Schramek D, Elling U
  TITLE     CRISPR-StAR enables high-resolution genetic screening in complex in 
            vivo models.
  JOURNAL   Nat Biotechnol. 2024 Dec 16. doi: 10.1038/s41587-024-02512-9.
  PUBMED    39681701
REFERENCE   2  (bases 1 to 8159)
  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 
            Biotechnol. 2024 Dec 16. doi: 10.1038/s41587-024-02512-9."
COMMENT     Sequence Label: CRISPR-StAR2 (Retro)
FEATURES             Location/Qualifiers
     source          1..8159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        309..512
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     463..483
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             513..688
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    751..950
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1151..1566
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1496..1518
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1534..1550
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1657..1905
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1657..1677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1828..1847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1947..1980)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2108..2141
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(2165..2198)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2687..3085
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(3094..3127)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3283..3316
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             3892..4695
                     /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(3955..3974)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4565..4584
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4866..5290
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5393..5415)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5551..5571)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5556..5885
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5736..5871
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5907..5926
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6007..6024)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6178..6766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6258..6277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6937..7728,7729..7797))
                     /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     7560..7579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7798..7902)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7970..7988
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(8088..8159,1..308)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      301 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      361 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      421 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      481 gtgtacggtg ggaggtctat ataagcagag ctcaataaaa gagcccacaa cccctcactc
      541 ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtattccca ataaagcctc
      601 ttgctgtttg catccgaatc gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
      661 tgactaccca cgacgggggt ctttcatttg ggggctcgtc cgggatttgg agacccctgc
      721 ccagggacca ccgacccacc accgggaggt aagctggcca gcaacttatc tgtgtctgtc
      781 cgattgtcta gtgtctatgt ttgatgttat gcgcctgcgt ctgtactagt tagctaacta
      841 gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg gccgcaaccc
      901 tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag gaagggagtc
      961 gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga acctaaaaca
     1021 gttcccgcct ccgtctgaat ttttgctttc ggtttggaac cgaagccgcg cgtcttgtct
     1081 gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc
     1141 tgaaaattag ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga
     1201 tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt ggttaccttc
     1261 tgctctgcag aatggccaac ctttaacgtc ggatggccgc gagacggcac ctttaaccga
     1321 gacctcatca cccaggttaa gatcaaggtc ttttcacctg gcccgcatgg acacccagac
     1381 caggtcccct acatcgtgac ctgggaagcc ttggcttttg acccccctcc ctgggtcaag
     1441 ccctttgtac accctaagcc tccgcctcct cttcctccat ccgccccgtc tctccccctt
     1501 gaacctcctc gttcgacccc gcctcgatcc tccctttatc cagccctcac tccttctcta
     1561 ggcgccggaa ttgaagatct ccgcggttaa ttaaaatgat acggcgacca ccgagatctg
     1621 tcttgatcta ggagctactc atcaacggtc acatgtgagg gcctatttcc catgattcct
     1681 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     1741 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1801 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1861 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc ttcgctcgag
     1921 agaagacctg tttaagagct atgctgataa cttcgtatag catacattat acgaagttat
     1981 tttttttttt tgcggccgcc aagagagcag aaatagcaag taactcatta ggtaaataag
     2041 gtagtccgtt tagtgccgta gccaattctc agacatttaa tggctctaaa cgtaacattt
     2101 acaaattata acttcgtata atgtgtacta tacgaagtta ttaattaatt ctggttcgaa
     2161 cctgataact tcgtatagca tacattatac gaagttatca ttcgaatgta gaattctacc
     2221 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     2281 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     2341 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     2401 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     2461 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     2521 ggggcagcgg ccaatagcag ctttgctcct tcgctttccg ggctcagggg cggggcgggc
     2581 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     2641 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gccaatatgg ccaagccttt
     2701 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
     2761 ctctgaggat tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
     2821 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
     2881 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     2941 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
     3001 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     3061 ctctggttat gtgtgggagg gctgacctgc aggataactt cgtatagcat acattatacg
     3121 aagttatcag catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca
     3181 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     3241 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gcataacttc gtataatgtg
     3301 tactatacga agttatctag aggtacccgt taagatcgga agagcacacg tctgaactcc
     3361 agtcacaaaa ttatctcgta tgccgtctta tgcttgttaa ttaacaattg acgcgtaatt
     3421 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     3481 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     3541 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     3601 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     3661 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     3721 cctttggggc agcggccaat agcagctttg ctccttcgct ttccgggctc aggggcgggg
     3781 cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg
     3841 ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctgcagccaa tatgggatcg
     3901 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3961 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4021 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4081 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4141 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4201 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4261 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4321 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4381 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4441 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4501 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4561 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4621 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4681 gacgagttct tctgagggga tccgtcgacc tgcagccaag cttatcgata ccgtcgagat
     4741 ttaattaaga tatcatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg
     4801 aagcctatag agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag
     4861 gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc
     4921 aaggcatgga aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat
     4981 ggaacagggt cgaccctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa
     5041 tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct
     5101 tctgctcccc gagctcaata aaagagccca caacccctca ctcggggcgc cagtcctccg
     5161 attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac
     5221 ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc cacgacgggg
     5281 gtctttcatt tgggggctcg tccgggattt ggagacccct gcccagggac caccgaccca
     5341 ccaccgggag gtaagctggc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     5401 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     5461 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     5521 gtagcgatag cggagtgtag atccggctgt ggaatgtgtg tcagttaggg tgtggaaagt
     5581 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5641 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5701 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5761 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5821 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5881 gcaaaaagct tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     5941 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     6001 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6061 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6121 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6181 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6241 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6301 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6361 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6421 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6481 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6541 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6601 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6661 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6721 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6781 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6841 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6901 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6961 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7021 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7081 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7141 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7201 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     7261 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7321 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7381 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7441 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7501 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     7561 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7621 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7681 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7741 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7801 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7861 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7921 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7981 cacgaggccc tttcgtgttc aagaattagc ttggccattg catacgttgt atccatatca
     8041 taatatgtac atttatattg gctcatgtcc aacattaccg ccatgttgac attgattatt
     8101 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagt
//
