LOCUS       sequence-383990-        7450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA in a third generation gRNA backbone with mCherry, targeting 
            region immediately downstream of MDM4, to be used with 
            MDM4_Deletion_Upstream_gRNA_1/2 for MDM4 deletion.
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_Deletion_Downstream_gRNA_2
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..43)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(23..41)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(62..78)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(62..78)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(75..97)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    86..102
                     /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)."
     promoter        complement(join(110..116,117..134,135..140))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    155..176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(293..310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(464..1052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(544..563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1223..2014,2015..2083))
                     /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     1846..1865
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2084..2188)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2214..2669)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2351..2372)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2563..2582
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2796..2818
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2810..2827
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2811..2827
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2837..2856
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2837..2855
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2876..3011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3051..3172
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3062..3081)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3116..3135
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             3250..3483
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3555..4143
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3661..3672
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4070..4090
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4159..4869)
                     /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(4437..4456)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4706..4724
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(4870..4893)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4893..4902
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4922..5133)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    5202..5319
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(5485..5733)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5543..5562)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5713..5733)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(5911..5955)
                     /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"
     misc_feature    6140..6373
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6866..6991
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             7038..7218
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(7219..7445)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 cttgcagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
       61 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
      121 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
      181 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
      241 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
      301 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      361 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      421 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      481 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      541 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
      601 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
      661 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
      721 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      781 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      841 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      901 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      961 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1021 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1081 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     1141 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1201 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1261 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1321 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1381 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1441 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1501 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     1561 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     1621 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1681 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1741 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1801 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1861 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1921 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1981 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     2041 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2101 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2161 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     2221 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2281 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2341 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2401 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2461 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     2521 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     2581 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     2641 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     2701 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     2761 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     2821 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtacgt ccctaggcct
     2881 ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct
     2941 gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt
     3001 aggggcggga tgggcggagt taggggcggg atagctagag ccagacatga taagatacat
     3061 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3121 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttcctctcac
     3181 tctctgatat tcatttcttt gcaagttata aatactgaat aataagatga catgaactac
     3241 tatacgtact gctagagatt ttccacactg actaaaaggg tctgagggat ctctagttac
     3301 cagagtcaca caacagacgg gcacacacta cttgaagcac tcaaggcaag ctttattgag
     3361 gcttaagcag tgggttccct agttagccag agagctccca ggctcagatc tggtctaacc
     3421 agagagaccc agtacaagca aaaagcagat cttgtcttcg ttgggagtga attagccctt
     3481 ccagtccccc cttttctttt aaaaagtggc taagatctac agctgccttg taagtcattg
     3541 gtcttaaagt cgacgcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag
     3601 gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg
     3661 attgagggcc gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca
     3721 ggcgagcagc caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc
     3781 caacagccga gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc
     3841 aatagggagg gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca
     3901 accagtgggg gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg
     3961 ccacaactcc tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc
     4021 catacgggaa gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa
     4081 aggagcaaca tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg
     4141 attgtcgact taacgcgtct acttgtacag ctcgtccatg ccgccggtgg agtggcggcc
     4201 ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa
     4261 cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt
     4321 cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc
     4381 cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt
     4441 cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat
     4501 gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
     4561 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat
     4621 gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc
     4681 ccaggcgaag ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc
     4741 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
     4801 catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt
     4861 gctcaccatc ttatcgtcat cgtctttgta atccatggtg gcggatcccg cgtcacgaca
     4921 cctgtgttct ggcggcaaac ccgttgcgaa aaagaacgtt cacggcgact actgcactta
     4981 tatacggttc tcccccaccc tcgggaaaaa ggcggagcca gtacacgaca tcactttccc
     5041 agtttacccc gcgccacctt ctctaggcac cggttcaatt gccgacccct ccccccaact
     5101 tctcggggac tgtgggcgat gtgcgctctg cccactgacg ggcaccggag ccactcgagc
     5161 cggcgccaaa gtggatctct gctgtccctg taataaaccc gaaaattttg aatttttgta
     5221 atttgttttt gtaattcttt agtttgtatg tctgttgcta ttatgtctac tattctttcc
     5281 cctgcactgt accccccaat cccccctttt cttttaaaat tgtggatgaa tactgccatt
     5341 tgtctcaaga tctagaattc aaaaaaagca ccgactcggt gccactttgc tgtttccagc
     5401 aaagttgata acggactagc cttatttaaa cttgctatgc tgtttccagc atagctctta
     5461 aaccgagact atctgtaacc tagcggtgtt tcgtcctttc cacaagatat ataaagccaa
     5521 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
     5581 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
     5641 ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg
     5701 caaatatgaa ggaatcatgg gaaataggcc ctctctgggt cccctcgggg ttgggaggtg
     5761 ggtctgaaac gataatggtg aatatccctg cctaactcta ttcactatag aaagtacagc
     5821 aaaaactatt cttaaaccta ccaagcctcc tactatcatt atgaataatt ttatatacca
     5881 cagccaattt gttatgttaa accaattcca caaacttgcc catttatcta attccaataa
     5941 ttcttgttca ttcttttctt gctggttttg cgattcttca attaaggagt gtattaagct
     6001 tgtgtaattg ttaatttctc tgtcccactc catccaggtc gtgtgattcc aaatctgttc
     6061 cagagattta ttactccaac tagcattcca aggcacagca gtggtgcaaa tgagttttcc
     6121 agagcaaccc caaatcccca ggagctgttg atcctttagg tatctttcca cagccaggat
     6181 tcttgcctgg agctgcttga tgccccagac tgtgagttgc aacagatgct gttgcgcctc
     6241 aatagccctc agcaaattgt tctgctgctg cactatacca gacaataatt gtctggcctg
     6301 taccgtcagc gtcattgacg ctgcgcccat agtgcttcct gctgctccca agaacccaag
     6361 gaacaaagct cctattccca ctgctctttt ttctctctgc accactcttc tctttgcctt
     6421 ggtgggtgct actcctaatg gttcaatttt tactacttta tatttatata attcacttct
     6481 ccaattgtcc ctcatatctc ctcctccagg tctgaagatc agcggccgct tgctgtgcgg
     6541 tggtcttact tttgttttgc tcttcctcta tcttgtctaa agcttccttg gtgtctttta
     6601 tctctatcct ttgatgcaca caatagaggg ttgctactgt attatataat gatctaagtt
     6661 cttctgatcc tgtctgaagg gatggttgta gctgtcccag tatttgtcta cagccttctg
     6721 atgtttctaa caggccagga ttaactgcga atcgttctag ctccctgctt gcccatacta
     6781 tatgttttaa tttatatttt ttctttcccc ctggccttaa ccgaattttt tcccatcgcg
     6841 atctaattct cccccgctta atactgacgc tctcgcaccc atctctctcc ttctagcctc
     6901 cgctagtcaa aatttttggc gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg
     6961 cttcagcaag ccgagtcctg cgtcgagaga gctcctctgg tttccctttc gctttcaagt
     7021 ccctgttcgg gcgccactgc tagagatttt ccacactgac taaaagggtc tgagggatct
     7081 ctagttacca gagtcacaca acagacgggc acacactact tgaagcactc aaggcaagct
     7141 ttattgaggc ttaagcagtg ggttccctag ttagccagag agctcccagg ctcagatctg
     7201 gtctaaccag agagacccgt ttatgtatcg agctaggcac ttaaatacaa tatctctgca
     7261 atgcggcaat tcagtggttc gtccaatcca tgtcagaccc gtctgttgcc ttcctaataa
     7321 ggcacgatcg taccacctta cttccaccaa tcggcatgca cggtgctttt tctctccttg
     7381 taaggcatgt tgctaactca tcgttaccat gttgcaagac tacaagagta ttgcataaga
     7441 ctacattaag
//
