LOCUS       sequence-182971-        7301 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 7301)
  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 7301)
  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 H10.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    105..126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(141..146,147..164,165..171)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    179..195
                     /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     184..206
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     203..219
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     203..219
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     238..258
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        240..258
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        286..512
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             513..693
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    740..865
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1358..1591
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1776..1820
                     /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        1998..2246
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1998..2018
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2169..2188
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2401..2518
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2587..2798
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2824..3423
                     /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(2824..2843)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3320..3340
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3439..4027
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3492..3512)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3910..3921)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4099..4332
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4410..4531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4447..4466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4501..4520
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4571..4706
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4726..4745)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4727..4745)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4755..4772)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4755..4771)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4764..4786)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4913..5368
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5000..5019)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5210..5231
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5394..5498
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5499..5567,5568..6359)
                     /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(5717..5736)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6530..7118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7019..7038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7272..7289
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
       61 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      121 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      181 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      241 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
      301 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      361 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      421 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      481 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      541 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      601 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      661 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      721 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      781 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      841 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      901 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      961 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     1021 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1081 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1141 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1201 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1261 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1321 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1381 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1441 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1501 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1561 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1621 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1681 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1741 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1801 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1861 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1921 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     1981 ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2041 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     2101 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2161 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2221 atatcttgtg gaaaggacga aacaccgcgc ttcccccaga taaatagttt aagagctatg
     2281 ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca
     2341 ccgagtcggt gctttttttg aattctagat cttgagacaa atggcagtat tcatccacaa
     2401 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2461 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2521 ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc
     2581 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2641 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2701 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2761 acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc
     2821 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     2881 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     2941 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3001 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3061 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3121 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3181 gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg
     3241 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3301 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3361 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3421 tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3481 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3541 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3601 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3661 gctgactcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3721 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3781 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3841 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     3901 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3961 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     4021 tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     4081 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     4141 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     4201 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     4261 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     4321 aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg
     4381 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     4441 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4501 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     4561 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4621 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4681 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     4741 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4801 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4861 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     4921 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4981 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5041 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5101 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5161 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5221 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5281 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5341 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     5401 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5461 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5521 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5581 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5641 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5701 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5761 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5821 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5881 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5941 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6001 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6061 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6121 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6181 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6241 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6301 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6361 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6421 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6481 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6541 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6601 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6661 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6721 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6781 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6841 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6901 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6961 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7021 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7081 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7141 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7201 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     7261 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga g
//
