LOCUS       sequence-182957-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SKA1.
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: SKA1 H3.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    102..123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(138..143,144..161,162..168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    176..192
                     /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     181..203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     200..216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     200..216
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     235..255
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        237..255
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        283..509
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             510..690
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    737..862
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1355..1588
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1773..1817
                     /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        1995..2243
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1995..2015
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2166..2185
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2399..2516
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2585..2796
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2822..3421
                     /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(2822..2841)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3318..3338
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3437..4025
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3490..3510)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3908..3919)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4097..4330
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4408..4529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4445..4464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4499..4518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4569..4704
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4724..4743)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4725..4743)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4753..4770)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4753..4769)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4762..4784)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4911..5366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4998..5017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5208..5229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5392..5496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5497..5565,5566..6357)
                     /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(5715..5734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6528..7116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7017..7036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7270..7287
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      121 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      181 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
      241 aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac
      301 tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa
      361 agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc
      421 aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg
      481 tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct
      541 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
      601 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
      661 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
      721 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
      781 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
      841 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
      901 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
      961 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     1021 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1081 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1141 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1201 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1261 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1321 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1381 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1441 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1501 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1561 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     1621 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     1681 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     1741 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     1801 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     1861 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     1921 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     1981 cgaggggacc cagagagggc ctatttccca tgattccttc atatttgcat atacgataca
     2041 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2101 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2161 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     2221 tcttgtggaa aggacgaaac accgtaattg ttccagatct gacggtttaa gagctatgct
     2281 ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc
     2341 gagtcggtgc tttttttgaa ttctagatct tgagacaaat ggcagtattc atccacaatt
     2401 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2461 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2521 tttattacag ggacagcaga gatccacttt ggcgccggct cgagtggctc cggtgcccgt
     2581 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2641 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2701 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2761 gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac
     2821 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     2881 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     2941 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     3001 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     3061 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3121 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3181 gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg
     3241 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3301 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3361 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     3421 aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3481 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3541 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3601 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3661 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     3721 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     3781 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     3841 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     3901 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     3961 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4021 cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     4081 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     4141 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     4201 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     4261 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     4321 atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca
     4381 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
     4441 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4501 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     4561 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4621 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4681 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
     4741 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4801 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4861 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     4921 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4981 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5041 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5101 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5161 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5221 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5281 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5341 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5401 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5461 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5521 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5581 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5641 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5701 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5761 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5821 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5881 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5941 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6001 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6061 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6121 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6181 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6241 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6301 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6361 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6421 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6481 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6541 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6601 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6661 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6721 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6781 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6841 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6901 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6961 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7021 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7081 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7141 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7201 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7261 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gc
//
