LOCUS       sequence-183239-        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 B3.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..20
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    137..158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(173..178,179..196,197..203)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    211..227
                     /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     216..238
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     235..251
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     235..251
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     270..290
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        272..290
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        318..544
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             545..725
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    772..897
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1390..1623
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1808..1852
                     /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        2030..2278
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2030..2050
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2201..2220
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2434..2551
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2620..2831
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2857..3456
                     /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(2857..2876)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3353..3373
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3472..4060
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3525..3545)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3943..3954)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4132..4365
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4443..4564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4480..4499)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4534..4553
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4604..4739
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4759..4778)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4760..4778)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4788..4805)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4788..4804)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4797..4819)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4946..5401
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5033..5052)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5243..5264
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5427..5531
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5532..5600,5601..6392)
                     /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(5750..5769)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6563..7151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7052..7071
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
       61 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      121 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      181 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      241 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
      301 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
      361 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
      421 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
      481 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
      541 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      601 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      661 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      721 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
      781 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
      841 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
      901 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
      961 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     1021 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1081 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1141 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1201 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     1261 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     1321 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     1381 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     1441 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     1501 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     1561 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     1621 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     1681 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     1741 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     1801 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     1861 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     1921 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     1981 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     2041 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     2101 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2161 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2221 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc
     2281 gccaacgcgc tcggcgtagg tttaagagct atgctggaaa cagcatagca agtttaaata
     2341 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta
     2401 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2581 actttggcgc cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2641 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2701 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2761 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2821 ccagaacaca ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa gcccacggtg
     2881 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     2941 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     3001 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3061 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3121 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3181 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3241 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3301 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3361 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     3421 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
     3481 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     3541 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     3601 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     3661 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3721 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3781 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     3841 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     3901 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     3961 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     4021 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     4081 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4141 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     4201 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     4261 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4321 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
     4381 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     4441 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4501 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4561 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     4621 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4681 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4741 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4801 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4861 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4921 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4981 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5041 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5101 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5161 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5221 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5281 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5341 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     5401 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5461 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5521 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5581 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5641 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5701 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5761 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5821 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5881 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5941 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6001 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6061 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6121 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6181 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6241 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6301 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6361 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6421 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6481 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6541 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6601 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6661 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6721 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6781 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6841 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6901 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6961 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7021 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7081 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7141 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7201 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     7261 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gc
//
