LOCUS       sequence-183577-        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 A11.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..27
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    144..165
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(180..185,186..203,204..210)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    218..234
                     /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     223..245
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     242..258
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     242..258
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     277..297
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        279..297
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        325..551
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             552..732
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    779..904
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1397..1630
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1815..1859
                     /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        2037..2285
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2037..2057
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2208..2227
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2441..2558
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2627..2838
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2864..3463
                     /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(2864..2883)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3360..3380
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3479..4067
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3532..3552)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3950..3961)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4139..4372
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4450..4571
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4487..4506)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4541..4560
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4611..4746
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4766..4785)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4767..4785)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4795..4812)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4795..4811)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4804..4826)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4953..5408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5040..5059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5250..5271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5434..5538
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5539..5607,5608..6399)
                     /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(5757..5776)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6570..7158
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7059..7078
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
       61 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
      121 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
      181 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      241 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
      301 caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca
      361 tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat
      421 tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat
      481 ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc
      541 gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
      601 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
      661 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
      721 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
      781 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
      841 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
      901 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
      961 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     1021 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     1081 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     1141 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     1201 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac
     1261 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     1321 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     1381 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     1441 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     1501 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     1561 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     1621 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     1681 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     1741 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     1801 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     1861 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     1921 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     1981 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccagagagg
     2041 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2101 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2161 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2221 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     2281 acaccgtata atcaattgga cgagacgttt aagagctatg ctggaaacag catagcaagt
     2341 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
     2401 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2461 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2521 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2581 gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca
     2641 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
     2701 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2761 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2821 tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatgaccg agtacaagcc
     2881 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2941 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     3001 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     3061 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3121 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3181 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3241 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3301 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3361 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3421 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     3481 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3541 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3601 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3661 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3721 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3781 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3841 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3901 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3961 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4021 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     4081 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4141 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4201 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4261 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4321 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     4381 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     4441 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4501 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4561 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     4621 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4681 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4741 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     4801 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4861 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4921 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     4981 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5041 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5101 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5161 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5221 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5281 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5341 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5401 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5461 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5521 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5581 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5641 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5701 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5761 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5821 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5881 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5941 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6001 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6061 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6121 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6181 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6241 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6301 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6361 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6421 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6481 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6541 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6601 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6661 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6721 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6781 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6841 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6901 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6961 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7021 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7081 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7141 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7201 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7261 accgcctttg agtgagctga taccgctcgc cgcagccgaa cg
//
