LOCUS       sequence-183232-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human KNTC1.
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: KNTC1 A4.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..25
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    142..163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(178..183,184..201,202..208)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    216..232
                     /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     221..243
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     240..256
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     240..256
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     275..295
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        277..295
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        323..549
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             550..730
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    777..902
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1395..1628
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1813..1857
                     /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        2035..2283
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2035..2055
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2206..2225
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2439..2556
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2625..2836
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2862..3461
                     /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(2862..2881)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3358..3378
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3477..4065
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3530..3550)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3948..3959)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4137..4370
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4448..4569
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4485..4504)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4539..4558
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4609..4744
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4764..4783)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4765..4783)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4793..4810)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4793..4809)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4802..4824)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4951..5406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5038..5057)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5248..5269
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5432..5536
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5537..5605,5606..6397)
                     /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(5755..5774)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6568..7156
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7057..7076
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
       61 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      121 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      181 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
      301 aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg
      361 gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg
      421 gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
      481 attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga
      541 tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
      601 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
      661 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
      721 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
      781 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
      841 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
      901 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
      961 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     1021 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     1081 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     1141 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     1201 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
     1261 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1321 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1381 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1441 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1501 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1561 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1621 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1681 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     1741 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     1801 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     1861 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     1921 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     1981 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cagagagggc
     2041 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2101 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2161 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2221 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     2281 accgaggcat gcagcttaag tgatgtttaa gagctatgct ggaaacagca tagcaagttt
     2341 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa
     2401 ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     2461 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2521 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     2581 gatccacttt ggcgccggct cgagtggctc cggtgcccgt cagtgggcag agcgcacatc
     2641 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2701 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2761 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2821 tgccgccaga acacaggtgt cgtgacgcgg gatccgccac catgaccgag tacaagccca
     2881 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     2941 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     3001 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3061 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     3121 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     3181 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     3241 ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     3301 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     3361 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     3421 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc
     3481 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3541 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3601 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3661 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3721 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3781 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3841 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3901 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3961 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     4021 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc
     4081 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     4141 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     4201 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4261 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4321 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag
     4381 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     4441 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4501 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4561 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta
     4621 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4681 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4741 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     4801 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4861 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4921 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     4981 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5041 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5101 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5161 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5221 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5281 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5341 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5401 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5461 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5521 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5581 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5641 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5701 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5761 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5821 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5881 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5941 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6001 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6061 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6121 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6181 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6241 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6301 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6361 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6421 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6481 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6541 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6601 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6661 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6721 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6781 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6841 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6901 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6961 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7021 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7081 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7141 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7201 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7261 cgcctttgag tgagctgata ccgctcgccg cagccgaacg ac
//
