LOCUS       sequence-183806-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human NEK2.
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: NEK2 F6.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..43
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    160..181
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(196..201,202..219,220..226)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    234..250
                     /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     239..261
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     258..274
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     258..274
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     293..313
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        295..313
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        341..567
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             568..748
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    795..920
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1413..1646
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1831..1875
                     /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        2053..2301
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2053..2073
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2224..2243
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2457..2574
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2643..2854
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2880..3479
                     /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(2880..2899)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3376..3396
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3495..4083
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3548..3568)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3966..3977)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4155..4388
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4466..4587
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4503..4522)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4557..4576
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4627..4762
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4782..4801)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4783..4801)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4811..4828)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4811..4827)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4820..4842)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4969..5424
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5056..5075)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5266..5287
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5450..5554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5555..5623,5624..6415)
                     /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(5773..5792)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6586..7174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7075..7094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
       61 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      121 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      181 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      241 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      301 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
      361 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
      421 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
      481 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
      541 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
      601 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
      661 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
      721 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
      781 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
      841 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
      901 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
      961 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     1021 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     1081 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     1141 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     1201 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     1261 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     1321 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     1381 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     1441 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     1501 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     1561 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     1621 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     1681 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     1741 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     1801 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     1861 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     1921 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     1981 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     2041 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2101 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     2161 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2221 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     2281 ttgtggaaag gacgaaacac cgtggtcata ccgtattgca tcgtttaaga gctatgctgg
     2341 aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     2401 gtcggtgctt tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt
     2461 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     2521 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     2581 tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg gtgcccgtca
     2641 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     2701 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     2761 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     2821 tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga tccgccacca
     2881 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     2941 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     3001 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     3061 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     3121 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     3181 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     3241 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     3301 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     3361 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     3421 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     3481 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     3541 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     3601 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3661 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3721 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     3781 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     3841 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     3901 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     3961 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     4021 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     4081 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     4141 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     4201 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4261 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4321 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4381 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     4441 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     4501 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4561 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     4621 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4681 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4741 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     4801 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4861 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4921 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     4981 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5041 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5101 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5161 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     5221 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5281 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     5341 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5401 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5461 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5521 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5581 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5641 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5701 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5761 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5821 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5881 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5941 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6001 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6061 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6121 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6181 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6241 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6301 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6361 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6421 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6481 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6541 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6601 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6661 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6721 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6781 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6841 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6901 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6961 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7021 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7081 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7141 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7201 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     7261 tgtggataac cgtattaccg cctttgagtg agctgatacc gc
//
