LOCUS       sequence-383988-        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_2
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(491..514)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      514..523
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(543..754)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    823..940
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(1106..1354)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1164..1183)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1334..1354)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1532..1576)
                     /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    1761..1994
                     /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    2487..2612
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2659..2839
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2840..3066)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3094..3114)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3094..3112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3133..3149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3133..3149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3146..3168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3157..3173
                     /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(3181..3187,3188..3205,3206..3211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3226..3247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3364..3381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3535..4123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3615..3634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4294..5085,5086..5154))
                     /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     4917..4936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5155..5259)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5285..5740)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5422..5443)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5634..5653
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5867..5889
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5881..5898
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5882..5898
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5908..5927
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5908..5926
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      5947..6082
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6122..6243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6133..6152)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6187..6206
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6321..6554
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6626..7214
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6732..6743
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7141..7161
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(7230..7459,1..484,485..487,488..490))
                     /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(7233..7459,1..484,485..487,488..490))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7275..7296)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcaccat cttatcgtca tcgtctttgt aatccatggt ggcggatccc gcgtcacgac
      541 acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt
      601 atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc
      661 cagtttaccc cgcgccacct tctctaggca ccggttcaat tgccgacccc tccccccaac
      721 ttctcgggga ctgtgggcga tgtgcgctct gcccactgac gggcaccgga gccactcgag
      781 ccggcgccaa agtggatctc tgctgtccct gtaataaacc cgaaaatttt gaatttttgt
      841 aatttgtttt tgtaattctt tagtttgtat gtctgttgct attatgtcta ctattctttc
      901 ccctgcactg taccccccaa tccccccttt tcttttaaaa ttgtggatga atactgccat
      961 ttgtctcaag atctagaatt caaaaaaagc accgactcgg tgccactttg ctgtttccag
     1021 caaagttgat aacggactag ccttatttaa acttgctatg ctgtttccag catagctctt
     1081 aaacccggtt catgccgccc atgccggtgt ttcgtccttt ccacaagata tataaagcca
     1141 agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt
     1201 ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata
     1261 tctttgtgtt tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat
     1321 gcaaatatga aggaatcatg ggaaataggc cctctctggg tcccctcggg gttgggaggt
     1381 gggtctgaaa cgataatggt gaatatccct gcctaactct attcactata gaaagtacag
     1441 caaaaactat tcttaaacct accaagcctc ctactatcat tatgaataat tttatatacc
     1501 acagccaatt tgttatgtta aaccaattcc acaaacttgc ccatttatct aattccaata
     1561 attcttgttc attcttttct tgctggtttt gcgattcttc aattaaggag tgtattaagc
     1621 ttgtgtaatt gttaatttct ctgtcccact ccatccaggt cgtgtgattc caaatctgtt
     1681 ccagagattt attactccaa ctagcattcc aaggcacagc agtggtgcaa atgagttttc
     1741 cagagcaacc ccaaatcccc aggagctgtt gatcctttag gtatctttcc acagccagga
     1801 ttcttgcctg gagctgcttg atgccccaga ctgtgagttg caacagatgc tgttgcgcct
     1861 caatagccct cagcaaattg ttctgctgct gcactatacc agacaataat tgtctggcct
     1921 gtaccgtcag cgtcattgac gctgcgccca tagtgcttcc tgctgctccc aagaacccaa
     1981 ggaacaaagc tcctattccc actgctcttt tttctctctg caccactctt ctctttgcct
     2041 tggtgggtgc tactcctaat ggttcaattt ttactacttt atatttatat aattcacttc
     2101 tccaattgtc cctcatatct cctcctccag gtctgaagat cagcggccgc ttgctgtgcg
     2161 gtggtcttac ttttgttttg ctcttcctct atcttgtcta aagcttcctt ggtgtctttt
     2221 atctctatcc tttgatgcac acaatagagg gttgctactg tattatataa tgatctaagt
     2281 tcttctgatc ctgtctgaag ggatggttgt agctgtccca gtatttgtct acagccttct
     2341 gatgtttcta acaggccagg attaactgcg aatcgttcta gctccctgct tgcccatact
     2401 atatgtttta atttatattt tttctttccc cctggcctta accgaatttt ttcccatcgc
     2461 gatctaattc tcccccgctt aatactgacg ctctcgcacc catctctctc cttctagcct
     2521 ccgctagtca aaatttttgg cgtactcacc agtcgccgcc cctcgcctct tgccgtgcgc
     2581 gcttcagcaa gccgagtcct gcgtcgagag agctcctctg gtttcccttt cgctttcaag
     2641 tccctgttcg ggcgccactg ctagagattt tccacactga ctaaaagggt ctgagggatc
     2701 tctagttacc agagtcacac aacagacggg cacacactac ttgaagcact caaggcaagc
     2761 tttattgagg cttaagcagt gggttcccta gttagccaga gagctcccag gctcagatct
     2821 ggtctaacca gagagacccg tttatgtatc gagctaggca cttaaataca atatctctgc
     2881 aatgcggcaa ttcagtggtt cgtccaatcc atgtcagacc cgtctgttgc cttcctaata
     2941 aggcacgatc gtaccacctt acttccacca atcggcatgc acggtgcttt ttctctcctt
     3001 gtaaggcatg ttgctaactc atcgttacca tgttgcaaga ctacaagagt attgcataag
     3061 actacattaa gcttgcagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     3121 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3181 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3241 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3301 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3361 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3421 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3481 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3541 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3601 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3661 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3721 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3781 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3841 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3901 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3961 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4021 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4081 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4141 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4201 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4261 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4321 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4381 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4441 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4501 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4561 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4621 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4681 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4741 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4801 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4861 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4921 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4981 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5041 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5101 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5161 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5221 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5281 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5341 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5401 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5461 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5521 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5581 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5641 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5701 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     5761 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     5821 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     5881 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacg
     5941 tccctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     6001 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     6061 tgggcggagt taggggcggg atgggcggag ttaggggcgg gatagctaga gccagacatg
     6121 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     6181 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     6241 gttcctctca ctctctgata ttcatttctt tgcaagttat aaatactgaa taataagatg
     6301 acatgaacta ctatacgtac tgctagagat tttccacact gactaaaagg gtctgaggga
     6361 tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca ctcaaggcaa
     6421 gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc aggctcagat
     6481 ctggtctaac cagagagacc cagtacaagc aaaaagcaga tcttgtcttc gttgggagtg
     6541 aattagccct tccagtcccc ccttttcttt taaaaagtgg ctaagatcta cagctgcctt
     6601 gtaagtcatt ggtcttaaag tcgacgcggg gaggcggccc aaagggagat ccgactcgtc
     6661 tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc gcgggaagga
     6721 aggtccgctg gattgagggc cgaagggacg tagcagaagg acgtcccgcg cagaatccag
     6781 gtggcaacac aggcgagcag ccaaggaaag gacgatgatt tccccgacaa caccacggaa
     6841 ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg caaggcaggc ggcgatgagt
     6901 tccgccgtgg caatagggag ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg
     6961 gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag tgcacaccac gccacgttgc
     7021 ctgacaacgg gccacaactc ctcataaaga gacagcaacc aggatttata caaggaggag
     7081 aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc agcgtatcca
     7141 catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca aattttgtaa
     7201 tccagaggtt gattgtcgac ttaacgcgtc tacttgtaca gctcgtccat gccgagagtg
     7261 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     7321 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     7381 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     7441 tggcggatct tgaagttca
//
