LOCUS       sequence-183805-        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 F5.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    86..107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(122..127,128..145,146..152)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    160..176
                     /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     165..187
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     184..200
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     184..200
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     219..239
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        221..239
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        267..493
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             494..674
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    721..846
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1339..1572
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1757..1801
                     /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        1979..2227
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1979..1999
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2150..2169
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2383..2500
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2569..2780
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2806..3405
                     /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(2806..2825)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3302..3322
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3421..4009
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3474..3494)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3892..3903)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4081..4314
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4392..4513
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4429..4448)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4483..4502
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4553..4688
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4708..4727)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4709..4727)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4737..4754)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4737..4753)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4746..4768)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4895..5350
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4982..5001)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5192..5213
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5376..5480
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5481..5549,5550..6341)
                     /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(5699..5718)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6512..7100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7001..7020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7254..7271
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
       61 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      121 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
      181 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
      241 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
      301 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
      361 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
      421 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
      481 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
      541 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
      601 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
      661 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
      721 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
      781 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
      841 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
      901 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
      961 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1021 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1081 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1141 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     1201 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1261 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1321 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1381 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1441 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1501 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1561 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     1621 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     1681 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     1741 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     1801 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     1861 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     1921 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga
     1981 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     2041 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     2101 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     2161 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     2221 aaacaccgat attgtgaagg aggggatcgt ttaagagcta tgctggaaac agcatagcaa
     2281 gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     2341 tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2401 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2461 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2521 cagagatcca ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca
     2581 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     2641 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2701 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2761 ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag
     2821 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     2881 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     2941 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3001 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     3061 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     3121 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     3181 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     3241 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     3301 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     3361 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac
     3421 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3481 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3541 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3601 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3661 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3721 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3781 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3841 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3901 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3961 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     4021 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     4081 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     4141 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     4201 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     4261 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     4321 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     4381 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4441 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4501 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     4561 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4621 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4681 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     4741 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     4801 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     4861 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     4921 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4981 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5041 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5101 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5161 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5221 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5281 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5341 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5401 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5461 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5521 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5581 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5641 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5701 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5761 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5821 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5881 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5941 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6001 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6061 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6121 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6181 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6241 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6301 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6361 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6421 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6481 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6541 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6601 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6661 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6721 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6781 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6841 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6901 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6961 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7021 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7081 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7141 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7201 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7261 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gc
//
