LOCUS       sequence-183405-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SKA3.
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: SKA3 G6.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    83..104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(119..124,125..142,143..149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    157..173
                     /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     162..184
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     181..197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     181..197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     216..236
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        218..236
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        264..490
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             491..671
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    718..843
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1336..1569
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1754..1798
                     /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        1976..2224
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1976..1996
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2147..2166
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2380..2497
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2566..2777
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2803..3402
                     /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(2803..2822)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3299..3319
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3418..4006
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3471..3491)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3889..3900)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4078..4311
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4389..4510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4426..4445)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4480..4499
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4550..4685
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4705..4724)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4706..4724)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4734..4751)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4734..4750)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4743..4765)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4892..5347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4979..4998)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5189..5210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5373..5477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5478..5546,5547..6338)
                     /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(5696..5715)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6509..7097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6998..7017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7251..7268
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
       61 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      121 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      181 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      241 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
      301 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
      361 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
      421 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
      481 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      541 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      601 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      661 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      721 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
      781 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
      841 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
      901 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
      961 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     1021 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     1081 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     1141 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     1201 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     1261 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     1321 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     1381 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     1441 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     1501 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     1561 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     1621 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     1681 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     1741 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     1801 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     1861 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     1921 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg
     1981 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2041 tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2101 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2161 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2221 caccgataca actgcttgca acagggttta agagctatgc tggaaacagc atagcaagtt
     2281 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga
     2341 attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2401 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2461 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2521 agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca gagcgcacat
     2581 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
     2641 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2701 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2761 ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc
     2821 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     2881 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     2941 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     3001 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     3061 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     3121 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     3181 accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     3241 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     3301 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     3361 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat
     3421 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3481 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3541 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3601 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3661 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3721 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3781 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3841 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3901 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3961 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     4021 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     4081 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     4141 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     4201 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     4261 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
     4321 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     4381 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4441 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4501 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     4561 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4621 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4681 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     4741 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4801 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4861 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4921 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4981 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5041 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5101 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5161 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5221 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5281 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5341 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5401 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5461 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5521 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5581 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5641 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5701 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5761 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5821 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5881 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5941 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6001 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6061 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6121 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6181 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6241 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6301 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6361 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6421 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6481 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6541 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6601 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6661 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6721 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6781 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6841 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6901 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6961 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7021 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7081 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7141 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7201 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7261 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tc
//
