LOCUS       sequence-183238-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human H2AFX.
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: H2AFX B4.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    118..139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(154..159,160..177,178..184)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    192..208
                     /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     197..219
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     216..232
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     216..232
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     251..271
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        253..271
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        299..525
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             526..706
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    753..878
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1371..1604
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1789..1833
                     /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        2011..2259
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2011..2031
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2182..2201
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2415..2532
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2601..2812
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2838..3437
                     /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(2838..2857)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3334..3354
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3453..4041
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3506..3526)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3924..3935)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4113..4346
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4424..4545
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4461..4480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4515..4534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4585..4720
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4740..4759)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4741..4759)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4769..4786)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4769..4785)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4778..4800)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4927..5382
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5014..5033)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5224..5245
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5408..5512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5513..5581,5582..6373)
                     /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(5731..5750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6544..7132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7033..7052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7286..7302,1)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
       61 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      121 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      181 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
      241 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
      301 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
      361 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
      421 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
      481 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
      541 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
      601 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
      661 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
      721 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
      781 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
      841 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
      901 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
      961 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     1021 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     1081 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     1141 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     1201 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1261 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1321 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1381 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1441 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1501 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1561 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1621 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     1681 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     1741 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     1801 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     1861 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     1921 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     1981 acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat
     2041 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2101 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2161 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2221 ttcttggctt tatatatctt gtggaaagga cgaaacaccg tacgccgagc gcgttggcgc
     2281 gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
     2341 ttgaaaaagt ggcaccgagt cggtgctttt tttgaattct agatcttgag acaaatggca
     2401 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     2461 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2521 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag
     2581 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2641 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2701 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2761 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg
     2821 acgcgggatc cgccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     2881 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     2941 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     3001 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     3061 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     3121 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     3181 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc
     3241 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     3301 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     3361 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     3421 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
     3481 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3541 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3601 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3661 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3721 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3781 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3841 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     3901 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3961 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     4021 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
     4081 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     4141 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     4201 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     4261 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     4321 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     4381 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     4441 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4501 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     4561 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4621 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4681 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     4741 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     4801 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     4861 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4921 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4981 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5041 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5101 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5161 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5221 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5281 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5341 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     5401 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6541 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6601 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6661 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6721 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6781 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6841 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6901 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6961 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7021 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7081 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7141 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     7201 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     7261 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc ga
//
