LOCUS       sequence-183237-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human KIF2A.
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: KIF2A D7.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    116..137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(152..157,158..175,176..182)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    190..206
                     /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     195..217
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     214..230
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     214..230
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     249..269
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        251..269
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        297..523
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             524..704
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    751..876
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1369..1602
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1787..1831
                     /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        2009..2257
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2009..2029
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2180..2199
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2413..2530
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2599..2810
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2836..3435
                     /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(2836..2855)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3332..3352
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3451..4039
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3504..3524)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3922..3933)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4111..4344
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4422..4543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4459..4478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4513..4532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4583..4718
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4738..4757)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4739..4757)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4767..4784)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4767..4783)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4776..4798)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4925..5380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5012..5031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5222..5243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5406..5510
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5511..5579,5580..6371)
                     /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(5729..5748)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6542..7130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7031..7050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7284..7301
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
       61 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      121 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      181 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      241 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
      301 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
      361 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
      421 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
      481 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
      541 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
      601 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
      661 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
      721 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
      781 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
      841 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
      901 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
      961 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     1021 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1081 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1141 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1201 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1261 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1321 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1381 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1441 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1501 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1561 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     1621 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     1681 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     1741 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     1801 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     1861 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     1921 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     1981 ccacctccca accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt
     2041 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     2101 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2161 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2221 cttggcttta tatatcttgt ggaaaggacg aaacaccgtc ttcatcagga acaaggtcgt
     2281 ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt
     2341 gaaaaagtgg caccgagtcg gtgctttttt tgaattctag atcttgagac aaatggcagt
     2401 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2461 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2521 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagtg
     2581 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2641 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2701 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2761 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac
     2821 gcgggatccg ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     2881 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     2941 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     3001 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     3061 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     3121 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     3181 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     3241 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     3301 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     3361 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     3421 aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa
     3481 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3541 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3601 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3661 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3721 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3781 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3841 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3901 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3961 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4021 ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc
     4081 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     4141 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     4201 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     4261 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     4321 agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt
     4381 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     4441 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4501 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     4561 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4621 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4681 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     4741 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     4801 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4861 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4921 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4981 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5041 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5101 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5161 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5221 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5281 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5341 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     5401 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5461 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5521 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5581 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5641 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5701 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5761 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5821 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5881 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5941 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6001 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6061 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6121 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6181 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6241 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6301 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6361 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6421 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6481 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6541 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6601 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6661 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6721 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6781 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6841 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6901 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6961 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7021 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7081 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7141 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7201 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     7261 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga gg
//
