LOCUS       sequence-182973-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TTK.
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: TTK H9.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     464..483
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(509..720)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    789..906
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(1062..1310)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1120..1139)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1290..1310)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1488..1532)
                     /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"
     misc_feature    1717..1950
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2443..2568
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2615..2795
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(2796..3022)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3050..3070)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3050..3068)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3089..3105)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3089..3105)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3102..3124)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3113..3129
                     /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)."
     promoter        complement(join(3137..3143,3144..3161,3162..3167))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3182..3203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3320..3337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3491..4079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3571..3590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4250..5041,5042..5110))
                     /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     4873..4892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5111..5215)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5241..5696)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5378..5399)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5590..5609
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5823..5845
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5837..5854
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5838..5854
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5864..5883
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5864..5882
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      5903..6038
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6078..6199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6089..6108)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6143..6162
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6277..6510
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6582..7170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6688..6699
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7097..7117
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(7186..7302,1..483))
                     /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(7269..7289)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc
       61 gctgcccaga cccttgccct ggtggtcggg cgagactccg acggtggcca ggaaccacgc
      121 gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg
      181 ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac
      241 gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc acaccttgcc
      301 gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg
      361 gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt
      421 gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt
      481 catggtggcg gatcccgcgt cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa
      541 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
      601 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
      661 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
      721 actgacgggc accggagcca ctcgagccgg cgccaaagtg gatctctgct gtccctgtaa
      781 taaacccgaa aattttgaat ttttgtaatt tgtttttgta attctttagt ttgtatgtct
      841 gttgctatta tgtctactat tctttcccct gcactgtacc ccccaatccc cccttttctt
      901 ttaaaattgt ggatgaatac tgccatttgt ctcaagatct agaattcaaa aaaagcaccg
      961 actcggtgcc actttttcaa gttgataacg gactagcctt atttaaactt gctatgctgt
     1021 ttccagcata gctcttaaac tactgatgaa ctaagcttga cggtgtttcg tcctttccac
     1081 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
     1141 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
     1201 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca
     1261 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc tctgggtccc
     1321 ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat atccctgcct aactctattc
     1381 actatagaaa gtacagcaaa aactattctt aaacctacca agcctcctac tatcattatg
     1441 aataatttta tataccacag ccaatttgtt atgttaaacc aattccacaa acttgcccat
     1501 ttatctaatt ccaataattc ttgttcattc ttttcttgct ggttttgcga ttcttcaatt
     1561 aaggagtgta ttaagcttgt gtaattgtta atttctctgt cccactccat ccaggtcgtg
     1621 tgattccaaa tctgttccag agatttatta ctccaactag cattccaagg cacagcagtg
     1681 gtgcaaatga gttttccaga gcaaccccaa atccccagga gctgttgatc ctttaggtat
     1741 ctttccacag ccaggattct tgcctggagc tgcttgatgc cccagactgt gagttgcaac
     1801 agatgctgtt gcgcctcaat agccctcagc aaattgttct gctgctgcac tataccagac
     1861 aataattgtc tggcctgtac cgtcagcgtc attgacgctg cgcccatagt gcttcctgct
     1921 gctcccaaga acccaaggaa caaagctcct attcccactg ctcttttttc tctctgcacc
     1981 actcttctct ttgccttggt gggtgctact cctaatggtt caatttttac tactttatat
     2041 ttatataatt cacttctcca attgtccctc atatctcctc ctccaggtct gaagatcagc
     2101 ggccgcttgc tgtgcggtgg tcttactttt gttttgctct tcctctatct tgtctaaagc
     2161 ttccttggtg tcttttatct ctatcctttg atgcacacaa tagagggttg ctactgtatt
     2221 atataatgat ctaagttctt ctgatcctgt ctgaagggat ggttgtagct gtcccagtat
     2281 ttgtctacag ccttctgatg tttctaacag gccaggatta actgcgaatc gttctagctc
     2341 cctgcttgcc catactatat gttttaattt atattttttc tttccccctg gccttaaccg
     2401 aattttttcc catcgcgatc taattctccc ccgcttaata ctgacgctct cgcacccatc
     2461 tctctccttc tagcctccgc tagtcaaaat ttttggcgta ctcaccagtc gccgcccctc
     2521 gcctcttgcc gtgcgcgctt cagcaagccg agtcctgcgt cgagagagct cctctggttt
     2581 ccctttcgct ttcaagtccc tgttcgggcg ccactgctag agattttcca cactgactaa
     2641 aagggtctga gggatctcta gttaccagag tcacacaaca gacgggcaca cactacttga
     2701 agcactcaag gcaagcttta ttgaggctta agcagtgggt tccctagtta gccagagagc
     2761 tcccaggctc agatctggtc taaccagaga gacccgttta tgtatcgagc taggcactta
     2821 aatacaatat ctctgcaatg cggcaattca gtggttcgtc caatccatgt cagacccgtc
     2881 tgttgccttc ctaataaggc acgatcgtac caccttactt ccaccaatcg gcatgcacgg
     2941 tgctttttct ctccttgtaa ggcatgttgc taactcatcg ttaccatgtt gcaagactac
     3001 aagagtattg cataagacta cattaagctt gcagctccag cttttgttcc ctttagtgag
     3061 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3121 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3181 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3241 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3301 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3361 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3421 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3481 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3541 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3601 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3661 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3721 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3781 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3841 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3901 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3961 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4021 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4081 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4141 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4201 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4261 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4321 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4381 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4441 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4501 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4561 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4621 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4681 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4741 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4801 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4861 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4921 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4981 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5041 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5101 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5161 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5221 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5281 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5341 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5401 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5461 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5521 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5581 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5641 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     5701 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     5761 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     5821 ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata
     5881 gggcgaattg ggtacgtccc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     5941 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     6001 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggata
     6061 gctagagcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     6121 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     6181 aagctgcaat aaacaagttc ctctcactct ctgatattca tttctttgca agttataaat
     6241 actgaataat aagatgacat gaactactat acgtactgct agagattttc cacactgact
     6301 aaaagggtct gagggatctc tagttaccag agtcacacaa cagacgggca cacactactt
     6361 gaagcactca aggcaagctt tattgaggct taagcagtgg gttccctagt tagccagaga
     6421 gctcccaggc tcagatctgg tctaaccaga gagacccagt acaagcaaaa agcagatctt
     6481 gtcttcgttg ggagtgaatt agcccttcca gtcccccctt ttcttttaaa aagtggctaa
     6541 gatctacagc tgccttgtaa gtcattggtc ttaaagtcga cgcggggagg cggcccaaag
     6601 ggagatccga ctcgtctgag ggcgaaggcg aagacgcgga agaggccgca gagccggcag
     6661 caggccgcgg gaaggaaggt ccgctggatt gagggccgaa gggacgtagc agaaggacgt
     6721 cccgcgcaga atccaggtgg caacacaggc gagcagccaa ggaaaggacg atgatttccc
     6781 cgacaacacc acggaattgt cagtgcccaa cagccgagcc cctgtccagc agcgggcaag
     6841 gcaggcggcg atgagttccg ccgtggcaat agggaggggg aaagcgaaag tcccggaaag
     6901 gagctgacag gtggtggcaa tgccccaacc agtgggggtt gcgtcagcaa acacagtgca
     6961 caccacgcca cgttgcctga caacgggcca caactcctca taaagagaca gcaaccagga
     7021 tttatacaag gaggagaaaa tgaaagccat acgggaagca atagcatgat acaaaggcat
     7081 taaagcagcg tatccacata gcgtaaaagg agcaacatag ttaagaatac cagtcaatct
     7141 ttcacaaatt ttgtaatcca gaggttgatt gtcgacttaa cgcgttcagg caccgggctt
     7201 gcgggtcatg caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa
     7261 gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag gt
//
