LOCUS       sequence-183203-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human KNSTRN.
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: KNSTRN D12.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..61
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    178..199
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(214..219,220..237,238..244)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    252..268
                     /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     257..279
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     276..292
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     276..292
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     311..331
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        313..331
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        359..585
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             586..766
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    813..938
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1431..1664
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1849..1893
                     /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        2071..2319
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2071..2091
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2242..2261
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2474..2591
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2660..2871
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2897..3496
                     /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(2897..2916)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3393..3413
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3512..4100
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3565..3585)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3983..3994)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4172..4405
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4483..4604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4520..4539)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4574..4593
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4644..4779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4799..4818)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4800..4818)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4828..4845)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4828..4844)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4837..4859)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4986..5441
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5073..5092)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5283..5304
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5467..5571
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5572..5640,5641..6432)
                     /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(5790..5809)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6603..7191
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7092..7111
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
       61 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      121 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      181 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      241 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
      301 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
      361 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
      421 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
      481 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
      541 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
      601 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
      661 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
      721 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
      781 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
      841 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
      901 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
      961 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     1021 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     1081 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     1141 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     1201 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     1261 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1321 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1381 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1441 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1501 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1561 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1621 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1681 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     1741 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     1801 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     1861 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     1921 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     1981 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     2041 acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat
     2101 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2161 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2221 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2281 ttcttggctt tatatatctt gtggaaagga cgaaacaccg aagtttcggg acaactgttg
     2341 tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact
     2401 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga caaatggcag
     2461 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2521 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2581 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagt
     2641 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     2701 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     2761 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     2821 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga
     2881 cgcgggatcc gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     2941 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     3001 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     3061 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     3121 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     3181 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     3241 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca
     3301 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     3361 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     3421 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     3481 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     3541 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3601 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3661 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3721 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3781 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3841 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3901 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3961 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     4021 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     4081 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     4141 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     4201 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     4261 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     4321 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     4381 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag
     4441 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     4501 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4561 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     4621 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4681 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4741 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     4801 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     4861 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4921 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4981 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5041 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5101 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5161 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5221 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5281 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5341 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5401 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     5461 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5521 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5581 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5641 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5701 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5761 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5821 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5881 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5941 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6001 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6061 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6121 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6181 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6241 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6301 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6361 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6421 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6481 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6541 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6601 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6661 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6721 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6781 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6841 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6901 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6961 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7021 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7081 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7141 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7201 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7261 gcgttatccc ctgattctgt ggataaccgt attaccgcct t
//
