LOCUS       sequence-441453-        8084 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 8084)
  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 8084)
  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-StAR4 (Retro)
FEATURES             Location/Qualifiers
     source          1..8084
                     /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)."
     CDS             2641..3039
                     /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(3048..3081)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3161..3194
                     /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             3817..4620
                     /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(3880..3899)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4490..4509
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4791..5215
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5318..5340)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5476..5496)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5481..5810
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5661..5796
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5832..5851
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5932..5949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6103..6691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6183..6202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6862..7653,7654..7722))
                     /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     7485..7504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7723..7827)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7895..7913
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(8013..8084,1..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 tgtagaattc taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     2221 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     2281 atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac
     2341 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     2401 aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa
     2461 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tccgggctca
     2521 ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc
     2581 gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcagccaat
     2641 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     2701 aacagcatcc ccatctctga ggattacagc gtcgccagcg cagctctctc tagcgacggc
     2761 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     2821 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     2881 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     2941 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     3001 cgtgaattgc tgccctctgg ttatgtgtgg gagggctgac ctgcaggata acttcgtata
     3061 gcatacatta tacgaagtta tcagcatagc aagtttaaat aaggctagtc cgttatcaac
     3121 ttgaaaaagt ggcaccgagt cggtgctttt ttgttttagc ataacttcgt ataatgtgta
     3181 ctatacgaag ttatctagag gtacccgtta agatcggaag agcacacgtc tgaactccag
     3241 tcacaaaatt atctcgtatg ccgtcttatg cttgttaatt aacaattgac gcgtaattct
     3301 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc
     3361 tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta
     3421 ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag
     3481 tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag
     3541 caydtmkmmy wrsccwggwr mgaaggrmma rmaccgctga gcaatggaag cgggtaggcc
     3601 tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa
     3661 ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggs gcccgaaggk
     3721 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct
     3781 cttcctcatc tccgggcctt tcgacctgca gccaatatgg gatcggccat tgaacaagat
     3841 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3901 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3961 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4021 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4081 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4141 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4201 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4261 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4321 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4381 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4441 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4501 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4561 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4621 ggggatccgt cgacctgcag ccaagcttat cgataccgtc gagatttaat taagatatca
     4681 tcagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac
     4741 gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     4801 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat
     4861 acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac agggtcgacc
     4921 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta
     4981 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
     5041 caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc
     5101 cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt
     5161 tccttgggag ggtctcctct gagtgattga ctacccacga cgggggtctt tcatttgggg
     5221 gctcgtccgg gatttggaga cccctgccca gggaccaccg acccaccacc gggaggtaag
     5281 ctggctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5341 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5401 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     5461 tgtagatccg gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     5521 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     5581 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     5641 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     5701 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     5761 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagcttactg
     5821 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     5881 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     5941 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6001 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6061 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6121 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6181 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6241 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6301 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6361 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6421 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6481 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6541 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6601 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6661 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6721 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6781 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6841 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6901 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6961 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7021 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7081 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7141 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     7201 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7261 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7321 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7381 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7441 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     7501 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7561 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7621 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7681 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7741 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7801 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7861 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7921 tgttcaagaa ttagcttggc cattgcatac gttgtatcca tatcataata tgtacattta
     7981 tattggctca tgtccaacat taccgccatg ttgacattga ttattgacta gttattaata
     8041 gtaatcaatt acggggtcat tagttcatag cccatatatg gagt
//
