LOCUS       sequence-383955-        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_1
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..233
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             234..414
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    461..586
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1079..1312
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1497..1541
                     /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        1719..1967
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1719..1739
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1890..1909
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2133..2250
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2319..2530
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2550..2559
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2559..2582
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2583..3293
                     /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(2728..2746)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2996..3015
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3309..3897
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3362..3382)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3780..3791)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3969..4202
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4280..4401
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4317..4336)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4371..4390
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4441..4576
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4596..4615)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4597..4615)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4625..4642)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4625..4641)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4634..4656)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4783..5238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4870..4889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5080..5101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5264..5368
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5369..5437,5438..6229)
                     /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(5587..5606)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6400..6988
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6889..6908
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7142..7159
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7276..7297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7312..7317,7318..7335,7336..7342)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7350..7366
                     /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     7355..7377
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7374..7390
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7374..7390
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7409..7429
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7411..7429
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
       61 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
      121 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
      181 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
      241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      421 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
      481 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
      541 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
      601 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
      661 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
      721 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
      781 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
      841 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
      901 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
      961 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     1021 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     1081 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     1141 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     1201 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     1261 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     1321 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     1381 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     1441 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     1501 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     1561 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     1621 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     1681 cgtttcagac ccacctccca accccgaggg gacccagaga gggcctattt cccatgattc
     1741 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     1801 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     1861 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     1921 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca gcttctgagg
     1981 ctctaccggt ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg
     2041 ttatcaactt tgctggaaac agcaaagtgg caccgagtcg gtgctttttt tgaattctag
     2101 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2161 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2221 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2281 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2341 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2401 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2461 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2521 cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata
     2581 agatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     2641 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2701 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     2761 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     2821 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2881 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2941 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     3001 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     3061 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     3121 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     3181 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     3241 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag tagacgcgtt
     3301 aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3361 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3421 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3481 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3541 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3601 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3661 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3721 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3781 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3841 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc
     3901 gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     3961 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     4021 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     4081 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     4141 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     4201 cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     4261 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     4321 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4381 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     4441 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4501 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4561 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     4621 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4681 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4741 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4801 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4861 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4921 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4981 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5041 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5101 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5161 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5221 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5281 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5341 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5401 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5461 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5521 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5581 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5641 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5701 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5761 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5821 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5881 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5941 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6001 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6061 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6121 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6181 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6241 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6301 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6361 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6421 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6481 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6541 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6601 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6661 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6721 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6781 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6841 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6901 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6961 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7021 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7081 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7141 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     7201 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7261 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7321 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7381 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     7441 ggagctgcaa
//
