LOCUS       sequence-183194-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human KIF4A.
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: KIF4A D9.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    122..143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(158..163,164..181,182..188)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    196..212
                     /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     201..223
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     220..236
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     220..236
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     255..275
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        257..275
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        303..529
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             530..710
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    757..882
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1375..1608
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1793..1837
                     /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        2015..2263
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2015..2035
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2186..2205
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2419..2536
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2605..2816
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2842..3441
                     /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(2842..2861)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3338..3358
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3457..4045
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3510..3530)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3928..3939)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4117..4350
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4428..4549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4465..4484)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4519..4538
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4589..4724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4744..4763)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4745..4763)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4773..4790)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4773..4789)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4782..4804)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4931..5386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5018..5037)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5228..5249
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5412..5516
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5517..5585,5586..6377)
                     /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(5735..5754)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6548..7136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7037..7056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7290..7302,1..5)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
       61 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
      121 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      181 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      241 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
      301 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
      361 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      421 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      481 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      541 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      601 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      661 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      721 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      781 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      841 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      901 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      961 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
     1021 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
     1081 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
     1141 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
     1201 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
     1261 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1321 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1381 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1441 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1501 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1561 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1621 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1681 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1741 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1801 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1861 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1921 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1981 tcagacccac ctcccaaccc cgaggggacc cagagagggc ctatttccca tgattccttc
     2041 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
     2101 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     2161 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     2221 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgagagaa tgaaccaaca
     2281 gttggtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat
     2341 caacttgaaa aagtggcacc gagtcggtgc tttttttgaa ttctagatct tgagacaaat
     2401 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     2461 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     2521 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct
     2581 cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     2641 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
     2701 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2761 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtgt
     2821 cgtgacgcgg gatccgccac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     2881 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     2941 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3001 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     3061 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     3121 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     3181 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac
     3241 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     3301 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     3361 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     3421 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt
     3481 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3541 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3601 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3661 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3721 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3781 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3841 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     3901 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3961 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     4021 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg
     4081 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     4141 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     4201 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     4261 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     4321 ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta
     4381 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     4441 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4501 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     4561 tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4621 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     4681 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat
     4741 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     4801 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     4861 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4921 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4981 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5041 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5101 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5161 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5221 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5281 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5341 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     5401 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5461 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5521 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5581 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5641 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5701 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5761 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5821 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5881 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5941 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6001 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6061 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6121 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6181 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6241 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6301 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6361 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6421 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6481 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6541 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6601 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6661 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6721 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6781 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6841 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6901 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6961 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7021 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7081 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7141 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7201 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     7261 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt ga
//
