LOCUS       sequence-441596-        8130 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 8130)
  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 8130)
  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-StAR3 (Retro)
FEATURES             Location/Qualifiers
     source          1..8130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..33
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        133..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             717..892
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    955..1154
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1355..1770
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1700..1722
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1738..1754
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1861..2109
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1861..1881
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2032..2051
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2151..2184)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2312..2345
                     /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(2369..2402)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2891..3289
                     /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(3298..3331)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3411..3444
                     /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)."
     promoter        3554..4053
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             4067..4870
                     /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(4130..4149)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4740..4759
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             5041..5465
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5568..5590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5726..5746)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5731..6060
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5911..6046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6082..6101
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6182..6199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6353..6941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6433..6452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7112..7903,7904..7972))
                     /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     7735..7754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7973..8077)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cattaaccta taaaaatagg cgtatcacga ggccctttcg tgttcaagaa ttagcttggc
       61 cattgcatac gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat
      121 taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcaat
      721 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc gtcgcccggg
      781 tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggtc tcgctgttcc
      841 ttgggagggt ctcctctgag tgattgacta cccacgacgg gggtctttca tttgggggct
      901 cgtccgggat ttggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
      961 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgtttgatg ttatgcgcct
     1021 gcgtctgtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
     1081 gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg
     1141 tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct
     1201 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
     1261 gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg
     1321 tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag
     1381 tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt
     1441 caagaagaga cgttggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg
     1501 ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca
     1561 cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct
     1621 tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct
     1681 ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt
     1741 tatccagccc tcactccttc tctaggcgcc ggaattgaag atctccgcgg ttaattaaaa
     1801 tgatacggcg accaccgaga tctgtcttga tctaggagct actcatcaac ggtcacatgt
     1861 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     1921 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     1981 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     2041 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     2101 cgaaacaccg ggtcttcgct cgagagaaga cctgtttaag agctatgctg ataacttcgt
     2161 atagcataca ttatacgaag ttattttttt tttttgcggc cgccaagaga gcagaaatag
     2221 caagtaactc attaggtaaa taaggtagtc cgtttagtgc cgtagccaat tctcagacat
     2281 ttaatggctc taaacgtaac atttacaaat tataacttcg tataatgtgt actatacgaa
     2341 gttattaatt aattctggtt cgaacctgat aacttcgtat agcatacatt atacgaagtt
     2401 atcattcgaa tgtagaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     2461 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     2521 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     2581 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     2641 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     2701 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     2761 tccgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     2821 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     2881 tgcagccaat atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     2941 ggctacaatc aacagcatcc ccatctctga ggattacagc gtcgccagcg cagctctctc
     3001 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     3061 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     3121 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     3181 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     3241 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctgac ctgcaggata
     3301 acttcgtata gcatacatta tacgaagtta tcagcatagc aagtttaaat aaggctagtc
     3361 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttagc ataacttcgt
     3421 ataatgtgta ctatacgaag ttatctagag gtacccgtta agatcggaag agcacacgtc
     3481 tgaactccag tcacaaaatt atctcgtatg ccgtcttatg cttgttaatt aacaattgac
     3541 gcgtaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     3601 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     3661 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     3721 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     3781 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     3841 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     3901 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     3961 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     4021 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gccaatatgg gatcggccat
     4081 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4141 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4201 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     4261 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4321 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4381 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4441 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4501 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4561 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4621 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4681 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4741 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4801 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4861 gttcttctga ggggatccgt cgacctgcag ccaagcttat cgataccgtc gagatttaat
     4921 taagatatca tcagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc
     4981 tatagagtac gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg
     5041 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
     5101 atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac
     5161 agggtcgacc ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     5221 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     5281 tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga
     5341 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     5401 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccacga cgggggtctt
     5461 tcatttgggg gctcgtccgg gatttggaga cccctgccca gggaccaccg acccaccacc
     5521 gggaggtaag ctggctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     5581 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     5641 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     5701 gatagcggag tgtagatccg gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     5761 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     5821 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     5881 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     5941 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     6001 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     6061 aagcttactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     6121 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     6181 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6241 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6301 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6361 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6421 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6481 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6541 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6601 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6661 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6721 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6781 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6841 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6901 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6961 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7021 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7081 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7141 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7201 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7261 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7321 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7381 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     7441 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7501 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7561 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7621 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7681 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     7741 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7801 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7861 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7921 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7981 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8041 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8101 ctgacgtcta agaaaccatt attatcatga
//
