LOCUS       sequence-183808-        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 A6.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    44..65
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(80..85,86..103,104..110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    118..134
                     /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     123..145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     142..158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     142..158
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     177..197
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        179..197
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        225..451
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             452..632
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    679..804
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1297..1530
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1715..1759
                     /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        1937..2185
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1937..1957
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2108..2127
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2341..2458
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2527..2738
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2764..3363
                     /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(2764..2783)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3260..3280
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3379..3967
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3432..3452)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3850..3861)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4039..4272
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4350..4471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4387..4406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4441..4460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4511..4646
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4666..4685)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4667..4685)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4695..4712)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4695..4711)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4704..4726)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4853..5308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4940..4959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5150..5171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5334..5438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5439..5507,5508..6299)
                     /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(5657..5676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6470..7058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6959..6978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7212..7229
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
       61 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      121 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
      181 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
      241 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
      301 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
      361 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
      421 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
      481 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
      541 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
      601 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
      661 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
      721 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
      781 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
      841 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
      901 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
      961 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1021 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     1081 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     1141 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     1201 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     1261 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     1321 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     1381 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     1441 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     1501 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     1561 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     1621 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     1681 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
     1741 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
     1801 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
     1861 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac
     1921 cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata
     1981 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     2041 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2101 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2161 tatcttgtgg aaaggacgaa acaccgctgt ctctggcaag taatccgttt aagagctatg
     2221 ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca
     2281 ccgagtcggt gctttttttg aattctagat cttgagacaa atggcagtat tcatccacaa
     2341 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2401 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2461 ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc
     2521 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2581 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2641 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2701 acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc
     2761 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     2821 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     2881 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     2941 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3001 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3061 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3121 gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg
     3181 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3241 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3301 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3361 tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3421 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3481 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3541 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3601 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3661 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3721 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3781 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     3841 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3901 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3961 tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     4021 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     4081 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     4141 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     4201 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     4261 aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg
     4321 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     4381 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4441 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     4501 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4561 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4621 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     4681 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4741 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4801 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     4861 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4921 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4981 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5041 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5101 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5161 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5221 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5281 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     5341 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5401 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5461 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5521 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5581 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5641 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5701 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5761 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5821 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5881 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5941 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6001 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6061 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6121 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6181 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6241 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6301 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6361 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6421 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6481 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6541 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6601 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6661 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6721 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6781 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6841 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6901 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6961 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7021 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7081 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7141 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     7201 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     7261 ctctccccgc gcgttggccg attcattaat gcagctggca cg
//
