LOCUS       sequence-183328-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CKAP5.
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: CKAP5 C5.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    119..140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(155..160,161..178,179..185)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    193..209
                     /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     198..220
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     217..233
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     217..233
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     252..272
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        254..272
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        300..526
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             527..707
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    754..879
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1372..1605
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1790..1834
                     /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        2012..2260
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2012..2032
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2183..2202
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2416..2533
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2602..2813
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2839..3438
                     /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(2839..2858)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3335..3355
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3454..4042
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3507..3527)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3925..3936)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4114..4347
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4425..4546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4462..4481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4516..4535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4586..4721
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4741..4760)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4742..4760)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4770..4787)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4770..4786)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4779..4801)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4928..5383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5015..5034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5225..5246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5409..5513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5514..5582,5583..6374)
                     /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(5732..5751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6545..7133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7034..7053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7287..7302,1..2)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
       61 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      121 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      181 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
      241 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
      301 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
      361 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
      421 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
      481 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt
      541 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
      601 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
      661 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
      721 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
      781 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
      841 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
      901 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
      961 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     1021 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1081 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1141 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1201 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     1261 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     1321 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     1381 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     1441 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     1501 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     1561 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     1621 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     1681 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     1741 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     1801 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     1861 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     1921 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     1981 gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata
     2041 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     2101 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2161 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2221 tttcttggct ttatatatct tgtggaaagg acgaaacacc gcctaaaaat ttggaccact
     2281 cgtttaagag ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa
     2341 cttgaaaaag tggcaccgag tcggtgcttt ttttgaattc tagatcttga gacaaatggc
     2401 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     2461 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     2521 aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga
     2581 gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     2641 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     2701 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     2761 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtgtcgt
     2821 gacgcgggat ccgccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     2881 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     2941 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     3001 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     3061 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     3121 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     3181 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac
     3241 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     3301 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     3361 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     3421 cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt
     3481 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3541 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3601 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3661 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3721 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3781 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3841 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     3901 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3961 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     4021 tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag
     4081 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     4141 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
     4201 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     4261 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     4321 tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc
     4381 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     4441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     4561 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4621 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4681 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     4741 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     4801 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     4861 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4921 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5341 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5521 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5581 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5641 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5701 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5761 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5821 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5881 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5941 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6001 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6061 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6121 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6181 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6241 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6301 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6361 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6421 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6481 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6541 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6601 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6661 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6721 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6781 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6841 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6901 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6961 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7021 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7081 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7141 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7201 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7261 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cg
//
