LOCUS       sequence-183307-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDC25A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the
            Diversity of p53-Dependent Responses to Cell-Cycle Defects.
  JOURNAL   Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 
            10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.
  PUBMED    28216383
REFERENCE   2  (bases 1 to 7302)
  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"; doi: 
            "10.1016/j.devcel.2017.01.012"; journalName: "Dev Cell"; date: 
            "2017-02-27- 27"; volume: "40"; issue: "4"; pages: "405-420.e2"
COMMENT     Sequence Label: CDC25A B1.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    90..111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(126..131,132..149,150..156)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    164..180
                     /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     169..191
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     188..204
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     188..204
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     223..243
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        225..243
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        271..497
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             498..678
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    725..850
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1343..1576
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1761..1805
                     /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        1983..2231
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1983..2003
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2154..2173
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2387..2504
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2573..2784
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2810..3409
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2810..2829)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3306..3326
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3425..4013
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3478..3498)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3896..3907)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4085..4318
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4396..4517
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4433..4452)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4487..4506
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4557..4692
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4712..4731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4713..4731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4741..4758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4741..4757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4750..4772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4899..5354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4986..5005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5196..5217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5380..5484
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5485..5553,5554..6345)
                     /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(5703..5722)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6516..7104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7005..7024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7258..7275
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
       61 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      121 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      181 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      241 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
      301 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
      361 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
      421 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
      481 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
      541 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
      601 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
      661 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
      721 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
      781 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
      841 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
      901 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
      961 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     1021 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     1081 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     1141 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
     1201 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     1261 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     1321 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     1381 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     1441 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     1501 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     1561 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     1621 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     1681 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     1741 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     1801 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     1861 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     1921 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca
     1981 gagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2041 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2101 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2161 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2221 gacgaaacac cgcccgtcgt gaaggcgcta ttgtttaaga gctatgctgg aaacagcata
     2281 gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     2341 tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2401 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2461 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2521 acagcagaga tccactttgg cgccggctcg agtggctccg gtgcccgtca gtgggcagag
     2581 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc
     2641 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     2701 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     2761 aacgggtttg ccgccagaac acaggtgtcg tgacgcggga tccgccacca tgaccgagta
     2821 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     2881 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     2941 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     3001 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     3061 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     3121 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     3181 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     3241 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     3301 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     3361 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt
     3421 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3481 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3541 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3601 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3661 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3721 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3781 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     3841 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     3901 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     3961 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4021 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4081 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     4141 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     4201 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     4261 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     4321 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4381 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4441 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4501 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     4561 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4621 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4681 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     4741 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4801 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4861 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     4921 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4981 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5041 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5101 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5161 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5221 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5281 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5341 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5401 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5461 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5521 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5581 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5641 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5701 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5761 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5821 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5881 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5941 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6001 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6061 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6121 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6181 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6241 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6301 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6361 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6421 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6481 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6601 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6661 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6721 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6781 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6841 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6901 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6961 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7021 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7081 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7141 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7201 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7261 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aa
//
