LOCUS       sequence-183305-        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 B2.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    113..134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(149..154,155..172,173..179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    187..203
                     /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     192..214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     211..227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     211..227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     246..266
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        248..266
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        294..520
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             521..701
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    748..873
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1366..1599
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1784..1828
                     /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        2006..2254
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2006..2026
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2177..2196
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2410..2527
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2596..2807
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2833..3432
                     /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(2833..2852)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3329..3349
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3448..4036
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3501..3521)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3919..3930)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4108..4341
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4419..4540
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4456..4475)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4510..4529
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4580..4715
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4735..4754)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4736..4754)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4764..4781)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4764..4780)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4773..4795)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4922..5377
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5009..5028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5219..5240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5403..5507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5508..5576,5577..6368)
                     /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(5726..5745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6539..7127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7028..7047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7281..7298
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
       61 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      121 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      181 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
      241 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
      301 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
      361 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
      421 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
      481 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
      541 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
      601 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
      661 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
      721 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
      781 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
      841 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
      901 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
      961 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     1021 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     1081 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     1141 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     1201 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     1261 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     1321 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     1381 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     1441 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     1501 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     1561 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     1621 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     1681 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     1741 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     1801 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     1861 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     1921 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     1981 cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca
     2041 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     2101 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2161 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2221 ggctttatat atcttgtgga aaggacgaaa caccgcgcgt cgcagcccgt cgtgagttta
     2281 agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa
     2341 aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa tggcagtatt
     2401 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2461 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2521 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     2581 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2641 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2701 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2761 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     2821 ggatccgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     2881 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     2941 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     3001 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     3061 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     3121 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3181 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     3241 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3301 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3361 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3421 cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga
     3481 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3541 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3601 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3661 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3721 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3781 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3841 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3901 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3961 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     4021 tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta
     4081 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     4141 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     4201 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     4261 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     4321 cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt
     4381 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata
     4441 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4501 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat
     4561 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4621 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4681 attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat
     4741 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4801 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     4861 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     4921 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4981 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5041 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5101 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5161 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5221 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5281 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5341 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     5401 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5461 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5521 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5581 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5641 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5701 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5761 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5821 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5881 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5941 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6001 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6061 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6121 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6181 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6241 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6301 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6361 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6421 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6481 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6541 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6601 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6661 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6721 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6781 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6841 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6901 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6961 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7021 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7081 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7141 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7201 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     7261 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga ag
//
