LOCUS       sequence-190027-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ACTL6A.
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: ACTL6A C8.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..19
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    136..157
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(172..177,178..195,196..202)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    210..226
                     /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     215..237
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     234..250
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     234..250
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     269..289
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        271..289
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        317..543
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             544..724
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    771..896
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1389..1622
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1807..1851
                     /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        2029..2277
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2029..2049
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2200..2219
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2433..2550
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2619..2830
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2856..3455
                     /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(2856..2875)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3352..3372
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3471..4059
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3524..3544)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3942..3953)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4131..4364
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4442..4563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4479..4498)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4533..4552
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4603..4738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4758..4777)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4759..4777)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4787..4804)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4787..4803)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4796..4818)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4945..5400
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5032..5051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5242..5263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5426..5530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5531..5599,5600..6391)
                     /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(5749..5768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6562..7150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7051..7070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
       61 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
      121 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
      181 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      241 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
      301 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
      361 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
      421 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
      481 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
      541 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      601 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      661 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
      721 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
      781 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
      841 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
      901 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
      961 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1021 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1081 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1141 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1201 agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga
     1261 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1321 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1381 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1441 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1501 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1561 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1621 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1681 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1741 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     1801 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     1861 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     1921 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     1981 tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga gggcctattt
     2041 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
     2101 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     2161 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     2221 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtc
     2281 ctacagctat tggtatgggt ttaagagcta tgctggaaac agcatagcaa gtttaaataa
     2341 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattctag
     2401 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2461 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2521 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2581 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2641 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2701 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2761 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2821 cagaacacag gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag cccacggtgc
     2881 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     2941 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     3001 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     3061 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     3121 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     3181 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     3241 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     3301 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     3361 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     3421 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
     3481 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3541 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3601 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3661 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3721 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3781 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3841 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     3901 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     3961 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4021 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     4081 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     4141 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     4201 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4261 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4321 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc
     4381 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     4441 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4501 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4561 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     4621 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4681 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4741 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     4801 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4861 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4921 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4981 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5041 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5101 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5161 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5221 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5281 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5341 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6241 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6541 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6601 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6661 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6721 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6781 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6841 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6901 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6961 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7021 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7081 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7141 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7201 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7261 tgagtgagct gataccgctc gccgcagccg aacgaccgag cg
//
