LOCUS       sequence-183807-        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 A5.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    109..130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(145..150,151..168,169..175)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    183..199
                     /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     188..210
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     207..223
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     207..223
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     242..262
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        244..262
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        290..516
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             517..697
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    744..869
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1362..1595
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1780..1824
                     /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        2002..2250
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2002..2022
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2173..2192
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2406..2523
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2592..2803
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2829..3428
                     /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(2829..2848)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3325..3345
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3444..4032
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3497..3517)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3915..3926)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4104..4337
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4415..4536
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4452..4471)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4506..4525
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4576..4711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4731..4750)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4732..4750)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4760..4777)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4760..4776)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4769..4791)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4918..5373
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5005..5024)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5215..5236
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5399..5503
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5504..5572,5573..6364)
                     /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(5722..5741)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6535..7123
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7024..7043
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7277..7294
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
       61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      121 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      181 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      241 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      301 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      361 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      421 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      481 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      541 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      601 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      661 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      721 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      781 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      841 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      901 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      961 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     1021 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1081 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1141 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1201 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1261 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1321 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1381 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1441 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1501 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1561 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1621 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1681 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1741 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1801 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1861 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1921 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1981 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     2041 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2101 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2161 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2221 ttatatatct tgtggaaagg acgaaacacc gctggaatcc tgcgattttt cgtttaagag
     2281 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2341 tggcaccgag tcggtgcttt ttttgaattc tagatcttga gacaaatggc agtattcatc
     2401 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2461 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2521 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga gtggctccgg
     2581 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     2641 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     2701 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     2761 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtgtcgt gacgcgggat
     2821 ccgccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     2881 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2941 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     3001 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     3061 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     3121 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     3181 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg
     3241 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     3301 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     3361 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     3421 gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     3481 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3541 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3601 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3661 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3721 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3781 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3841 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     3901 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3961 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4021 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     4081 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     4141 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4201 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4261 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4321 gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata
     4381 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     4441 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4501 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     4561 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4621 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4681 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     4741 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4801 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4861 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     4921 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4981 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5041 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5101 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5161 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5221 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5281 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5341 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     5401 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5461 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5521 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5581 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5641 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5701 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5761 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5821 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5881 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5941 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6001 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6061 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6121 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6181 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6241 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6301 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6361 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6421 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6481 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6541 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6601 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6661 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6721 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6781 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6841 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6901 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6961 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7021 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7081 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7141 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7201 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7261 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg ga
//
