LOCUS       sequence-383951-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TP53-targeting gRNA in the LRG2.1 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7459)
  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 7459)
  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: TP53-KO_gRNA_1
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     136..155
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     395..416
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(462..485)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      485..494
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(514..725)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    794..911
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(1077..1325)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1135..1154)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1305..1325)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1503..1547)
                     /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    1732..1965
                     /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    2458..2583
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2630..2810
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2811..3037)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3065..3085)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3065..3083)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3104..3120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3104..3120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3117..3139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3128..3144
                     /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(3152..3158,3159..3176,3177..3182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3197..3218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3335..3352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3506..4094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3586..3605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4265..5056,5057..5125))
                     /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     4888..4907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5126..5230)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5256..5711)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5393..5414)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5605..5624
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5838..5860
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5852..5869
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5853..5869
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5879..5898
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5879..5897
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      5918..6053
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6093..6214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6104..6123)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6158..6177
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6292..6525
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6597..7185
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6703..6714
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7112..7132
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(7201..7459,1..455,456..458,459..461))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(7204..7459,1..455,456..458,459..461))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7246..7267)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
       61 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
      121 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
      181 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
      241 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
      301 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
      361 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
      421 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tcttatcgtc atcgtctttg
      481 taatccatgg tggcggatcc cgcgtcacga cacctgtgtt ctggcggcaa acccgttgcg
      541 aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa
      601 aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc
      661 accggttcaa ttgccgaccc ctccccccaa cttctcgggg actgtgggcg atgtgcgctc
      721 tgcccactga cgggcaccgg agccactcga gccggcgcca aagtggatct ctgctgtccc
      781 tgtaataaac ccgaaaattt tgaatttttg taatttgttt ttgtaattct ttagtttgta
      841 tgtctgttgc tattatgtct actattcttt cccctgcact gtacccccca atcccccctt
      901 ttcttttaaa attgtggatg aatactgcca tttgtctcaa gatctagaat tcaaaaaaag
      961 caccgactcg gtgccacttt gctgtttcca gcaaagttga taacggacta gccttattta
     1021 aacttgctat gctgtttcca gcatagctct taaaccgtcc gcgccatggc catctcggtg
     1081 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     1141 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     1201 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcta
     1261 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     1321 ccctctctgg gtcccctcgg ggttgggagg tgggtctgaa acgataatgg tgaatatccc
     1381 tgcctaactc tattcactat agaaagtaca gcaaaaacta ttcttaaacc taccaagcct
     1441 cctactatca ttatgaataa ttttatatac cacagccaat ttgttatgtt aaaccaattc
     1501 cacaaacttg cccatttatc taattccaat aattcttgtt cattcttttc ttgctggttt
     1561 tgcgattctt caattaagga gtgtattaag cttgtgtaat tgttaatttc tctgtcccac
     1621 tccatccagg tcgtgtgatt ccaaatctgt tccagagatt tattactcca actagcattc
     1681 caaggcacag cagtggtgca aatgagtttt ccagagcaac cccaaatccc caggagctgt
     1741 tgatccttta ggtatctttc cacagccagg attcttgcct ggagctgctt gatgccccag
     1801 actgtgagtt gcaacagatg ctgttgcgcc tcaatagccc tcagcaaatt gttctgctgc
     1861 tgcactatac cagacaataa ttgtctggcc tgtaccgtca gcgtcattga cgctgcgccc
     1921 atagtgcttc ctgctgctcc caagaaccca aggaacaaag ctcctattcc cactgctctt
     1981 ttttctctct gcaccactct tctctttgcc ttggtgggtg ctactcctaa tggttcaatt
     2041 tttactactt tatatttata taattcactt ctccaattgt ccctcatatc tcctcctcca
     2101 ggtctgaaga tcagcggccg cttgctgtgc ggtggtctta cttttgtttt gctcttcctc
     2161 tatcttgtct aaagcttcct tggtgtcttt tatctctatc ctttgatgca cacaatagag
     2221 ggttgctact gtattatata atgatctaag ttcttctgat cctgtctgaa gggatggttg
     2281 tagctgtccc agtatttgtc tacagccttc tgatgtttct aacaggccag gattaactgc
     2341 gaatcgttct agctccctgc ttgcccatac tatatgtttt aatttatatt ttttctttcc
     2401 ccctggcctt aaccgaattt tttcccatcg cgatctaatt ctcccccgct taatactgac
     2461 gctctcgcac ccatctctct ccttctagcc tccgctagtc aaaatttttg gcgtactcac
     2521 cagtcgccgc ccctcgcctc ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga
     2581 gagctcctct ggtttccctt tcgctttcaa gtccctgttc gggcgccact gctagagatt
     2641 ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg
     2701 gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct
     2761 agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc gtttatgtat
     2821 cgagctaggc acttaaatac aatatctctg caatgcggca attcagtggt tcgtccaatc
     2881 catgtcagac ccgtctgttg ccttcctaat aaggcacgat cgtaccacct tacttccacc
     2941 aatcggcatg cacggtgctt tttctctcct tgtaaggcat gttgctaact catcgttacc
     3001 atgttgcaag actacaagag tattgcataa gactacatta agcttgcagc tccagctttt
     3061 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     3121 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3181 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3241 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3301 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3361 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3421 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3481 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3541 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3601 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3661 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3721 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3781 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3841 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3901 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3961 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4021 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4081 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4141 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4201 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4261 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4321 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4381 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4441 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4501 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4561 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4621 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4681 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4741 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4801 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4861 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4921 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4981 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5041 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5101 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5161 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5221 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     5281 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     5341 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     5401 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5461 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     5521 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     5581 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     5641 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     5701 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5761 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     5821 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
     5881 atacgactca ctatagggcg aattgggtac gtccctaggc ctccaaaaaa gcctcctcac
     5941 tacttctgga atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt
     6001 agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga
     6061 gttaggggcg ggatagctag agccagacat gataagatac attgatgagt ttggacaaac
     6121 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     6181 atttgtaacc attataagct gcaataaaca agttcctctc actctctgat attcatttct
     6241 ttgcaagtta taaatactga ataataagat gacatgaact actatacgta ctgctagaga
     6301 ttttccacac tgactaaaag ggtctgaggg atctctagtt accagagtca cacaacagac
     6361 gggcacacac tacttgaagc actcaaggca agctttattg aggcttaagc agtgggttcc
     6421 ctagttagcc agagagctcc caggctcaga tctggtctaa ccagagagac ccagtacaag
     6481 caaaaagcag atcttgtctt cgttgggagt gaattagccc ttccagtccc cccttttctt
     6541 ttaaaaagtg gctaagatct acagctgcct tgtaagtcat tggtcttaaa gtcgacgcgg
     6601 ggaggcggcc caaagggaga tccgactcgt ctgagggcga aggcgaagac gcggaagagg
     6661 ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct ggattgaggg ccgaagggac
     6721 gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca caggcgagca gccaaggaaa
     6781 ggacgatgat ttccccgaca acaccacgga attgtcagtg cccaacagcc gagcccctgt
     6841 ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg gcaataggga gggggaaagc
     6901 gaaagtcccg gaaaggagct gacaggtggt ggcaatgccc caaccagtgg gggttgcgtc
     6961 agcaaacaca gtgcacacca cgccacgttg cctgacaacg ggccacaact cctcataaag
     7021 agacagcaac caggatttat acaaggagga gaaaatgaaa gccatacggg aagcaatagc
     7081 atgatacaaa ggcattaaag cagcgtatcc acatagcgta aaaggagcaa catagttaag
     7141 aataccagtc aatctttcac aaattttgta atccagaggt tgattgtcga cttaacgcgt
     7201 ctacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag
     7261 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta
     7321 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc
     7381 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc
     7441 gttcttctgc ttgtcggcc
//
