LOCUS       sequence-441456-        8057 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 8057)
  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 8057)
  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-StAR1 (Retro)
FEATURES             Location/Qualifiers
     source          1..8057
                     /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    2008..2041
                     /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)."
     primer_bind     2457..2476
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2538..2936
                     /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(2945..2978)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3134..3167
                     /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             3790..4593
                     /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(3853..3872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4463..4482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4764..5188
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5291..5313)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5449..5469)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5454..5783
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5634..5769
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5805..5824
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5905..5922)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6076..6664)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6156..6175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6835..7626,7627..7695))
                     /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     7458..7477
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7696..7800)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7868..7886
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7986..8057,1..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 cagaattata acttcgtata atgtgtacta tacgaagtta
     2041 ttaattaatt ctggcctgca ggaattctac cgggtagggg aggcgctttt cccaaggcag
     2101 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     2161 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     2221 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     2281 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     2341 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     2401 ttcgctttcc gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt
     2461 ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct
     2521 ttcgacctgc agccaatatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     2581 gagcaacggc tacaatcaac agcatcccca tctctgagga ttacagcgtc gccagcgcag
     2641 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     2701 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     2761 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     2821 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     2881 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctgacctg
     2941 caggataact tcgtatagca tacattatac gaagttatca gcatagcaag tttaaataag
     3001 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
     3061 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
     3121 aaatggctct agcataactt cgtataatgt gtactatacg aagttatcta gaggtacccg
     3181 ttaagatcgg aagagcacac gtctgaactc cagtcacaaa attatctcgt atgccgtctt
     3241 atgcttgtta attaacaatt gacgcgtaat tctaccgggt aggggaggcg cttttcccaa
     3301 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     3361 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     3421 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     3481 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     3541 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     3601 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     3661 gcgggctcag gggcggggcg ggggccccaa gggccccccg aggccccgcc ttctgcacgc
     3721 ttcaaaaacg cacggctgcc gcgccgttct cctcttcctc atctcccggc ccttcgacct
     3781 gcagccaata tggggtcggc cattgaacaa gatggattgc acgcagggtc tccggccgct
     3841 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3901 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3961 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4021 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4081 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4141 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4201 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4261 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4321 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4381 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4441 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4501 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4561 gccttctatc gccttcttga cgagttcttc tgaggggatc cgtcgacctg cagccaagct
     4621 tatcgatacc gtcgagattt aattaagata tcatcagtgg tccaggctct agttttgact
     4681 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta
     4741 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     4801 taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga
     4861 tcaaggtcag gaacagatgg aacagggtcg accctagaga accatcagat gtttccaggg
     4921 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     4981 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     5041 cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct
     5101 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     5161 tgactaccca cgacgggggt ctttcatttg ggggctcgtc cgggatttgg agacccctgc
     5221 ccagggacca ccgacccacc accgggaggt aagctggctg cctcgcgcgt ttcggtgatg
     5281 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     5341 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     5401 cagccatgac ccagtcacgt agcgatagcg gagtgtagat ccggctgtgg aatgtgtgtc
     5461 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     5521 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5581 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     5641 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     5701 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     5761 ttggaggcct aggcttttgc aaaaagctta ctggcttaac tatgcggcat cagagcagat
     5821 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     5881 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5941 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6001 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6061 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6121 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6181 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6241 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6301 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6361 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6421 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6481 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     6541 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6601 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6661 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6721 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6781 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6841 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6901 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6961 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7021 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7081 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7141 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7201 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7261 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7321 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7381 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7441 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7501 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7561 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7621 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7681 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7741 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7801 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     7861 ctataaaaat aggcgtatca cgaggccctt tcgtgttcaa gaattagctt ggccattgca
     7921 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc
     7981 atgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     8041 tagcccatat atggagt
//
