LOCUS       sequence-183233-        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 A3.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     240..260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        288..514
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             515..695
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    742..867
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1360..1593
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1778..1822
                     /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        2000..2248
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2000..2020
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2171..2190
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2404..2521
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2590..2801
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2827..3426
                     /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(2827..2846)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3323..3343
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3442..4030
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3495..3515)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3913..3924)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4102..4335
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4413..4534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4450..4469)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4504..4523
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4574..4709
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4729..4748)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4730..4748)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4758..4775)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4758..4774)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4767..4789)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4916..5371
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5003..5022)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5213..5234
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5397..5501
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5502..5570,5571..6362)
                     /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(5720..5739)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6533..7121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7022..7041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7275..7292
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc
      301 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
      361 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
      421 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
      481 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
      541 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
      601 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
      661 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
      721 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
      781 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
      841 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
      901 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
      961 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
     1021 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     1081 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     1141 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     1201 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
     1261 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
     1321 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
     1381 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
     1441 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
     1501 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
     1561 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
     1621 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
     1681 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
     1741 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
     1801 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
     1861 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
     1921 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
     1981 aaccccgagg ggacccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2041 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     2101 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2161 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2221 atatatcttg tggaaaggac gaaacaccgc ttttcttgaa ccgacactcg tttaagagct
     2281 atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg
     2341 gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga caaatggcag tattcatcca
     2401 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2461 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2521 tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagt ggctccggtg
     2581 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     2641 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     2701 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     2761 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc
     2821 gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     2881 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     2941 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     3001 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     3061 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     3121 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     3181 aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt
     3241 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     3301 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     3361 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     3421 gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact
     3481 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3541 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3601 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3661 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3721 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3781 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3841 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3901 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3961 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4021 gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact
     4081 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc
     4141 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     4201 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     4261 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     4321 ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac
     4381 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     4441 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4501 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     4561 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4621 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4681 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     4741 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4801 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4861 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     4921 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4981 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5041 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5101 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     5161 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5221 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     5281 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5341 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     5401 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5461 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5521 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5581 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5641 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5701 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5761 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5821 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5881 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5941 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6001 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6061 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6121 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6181 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6241 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6301 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6361 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7141 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7201 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7261 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga ag
//
