LOCUS       sequence-383961-        7450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA from the Weissman CRISPRi library targeting MDM4, cloned into a
            third generation gRNA backbone with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7450)
  AUTHORS   Girish V, Lakhani AA, Thompson SL, Scaduto CM, Brown LM, Hagenson 
            RA, Sausville EL, Mendelson BE, Kandikuppa PK, Lukow DA, Yuan ML, 
            Stevens EC, Lee SN, Schukken KM, Akalu SM, Vasudevan A, Zou C, 
            Salovska B, Li W, Smith JC, Taylor AM, Martienssen RA, Liu Y, Sun R,
            Sheltzer JM
  TITLE     Oncogene-like addiction to aneuploidy in human cancers.
  JOURNAL   Science. 2023 Jul 6:eadg4521. doi: 10.1126/science.adg4521.
  PUBMED    37410869
REFERENCE   2  (bases 1 to 7450)
  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: "Science. 
            2023 Jul 6:eadg4521. doi: 10.1126/science.adg4521."
COMMENT     Sequence Label: MDM4_CRISPRi_gRNA_2
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1718..1966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1718..1738
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1889..1908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2132..2249
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2318..2529
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2549..2558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2558..2581
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2582..3292
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2727..2745)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2995..3014
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3308..3896
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3361..3381)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3779..3790)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3968..4201
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4279..4400
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4316..4335)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4370..4389
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4440..4575
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4595..4614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4596..4614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4624..4641)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4624..4640)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4633..4655)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4782..5237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4869..4888)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5079..5100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5263..5367
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5368..5436,5437..6228)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5586..5605)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6399..6987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6888..6907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7141..7158
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7275..7296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7311..7316,7317..7334,7335..7341)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7349..7365
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7354..7376
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7373..7389
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7373..7389
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7408..7428
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7410..7428
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     1741 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     1801 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     1861 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     1921 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcaa aatgctgccg
     1981 aggccctgtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     2041 tatcaacttt gctggaaaca gcaaagtggc accgagtcgg tgcttttttt gaattctaga
     2101 tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     2161 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     2221 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     2281 tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     2341 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     2401 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2461 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     2521 agaacacagg tgtcgtgacg cgggatccgc caccatggat tacaaagacg atgacgataa
     2581 gatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     2641 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     2701 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     2761 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     2821 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     2881 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     2941 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     3001 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     3061 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     3121 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     3181 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     3241 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt agacgcgtta
     3301 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3361 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3421 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3481 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3541 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3601 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3661 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3721 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3781 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3841 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     3901 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     3961 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     4021 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     4081 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     4141 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     4201 agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     4261 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4321 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4381 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     4441 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4501 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4561 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     4621 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4681 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4741 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     4801 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4861 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4921 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4981 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5041 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5101 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5161 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5221 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5281 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5341 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5401 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5461 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5521 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5581 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5641 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5701 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5761 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5821 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5881 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5941 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6001 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6061 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6121 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6181 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6241 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6301 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6361 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6421 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6481 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6541 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6601 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6661 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6721 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6781 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6841 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6901 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6961 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7021 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7081 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7141 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     7201 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     7261 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     7321 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     7381 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     7441 gagctgcaag
//
