LOCUS       sequence-190245-        7302 bp    DNA     circular SYN 15-APR-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 Apr 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 D10.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..70
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(85..90,91..108,109..115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    123..139
                     /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     128..150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     147..163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     147..163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     182..202
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        184..202
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        230..456
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             457..637
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    684..809
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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        1942..2190
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1942..1962
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2113..2132
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2346..2463
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2532..2743
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2769..3368
                     /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(2769..2788)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3265..3285
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3384..3972
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3437..3457)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3855..3866)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4044..4277
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4355..4476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4392..4411)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4446..4465
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4516..4651
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4671..4690)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4672..4690)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4700..4717)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4700..4716)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4709..4731)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4858..5313
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4945..4964)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5155..5176
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5339..5443
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5444..5512,5513..6304)
                     /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(5662..5681)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6475..7063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6964..6983
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7217..7234
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
       61 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      121 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      181 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      241 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      301 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      361 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      421 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      481 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      541 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      601 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      661 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      721 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      781 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      841 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      901 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      961 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1021 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1081 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1141 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     1981 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2041 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2101 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2161 ttatatatct tgtggaaagg acgaaacacc gatttgatcc ctctactgaa cgtttaagag
     2221 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2281 tggcaccgag tcggtgcttt ttttgaattc tagatcttga gacaaatggc agtattcatc
     2341 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2401 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2461 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga gtggctccgg
     2521 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     2581 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     2641 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     2701 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtgtcgt gacgcgggat
     2761 ccgccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     2821 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2881 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2941 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     3001 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     3061 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     3121 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg
     3181 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     3241 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     3301 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     3361 gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     3421 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3481 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3541 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3601 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3661 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3721 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3781 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     3841 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3901 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     3961 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     4021 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     4081 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4141 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4201 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4261 gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata
     4321 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     4381 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4441 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     4501 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4561 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4621 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     4681 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4741 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4801 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     4861 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4921 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4981 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5041 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5101 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5161 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5221 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5281 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     5341 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5401 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5461 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5521 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5581 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5641 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5701 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5761 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5821 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5881 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5941 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6001 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6061 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6121 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6181 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6241 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6301 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6361 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6421 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6481 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6541 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6601 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6661 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6721 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6781 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6841 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6901 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6961 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7021 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7081 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7141 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7201 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7261 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tg
//
