LOCUS       sequence-183432-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human BRCA2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  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 7301)
  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: BRCA2 A11.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    85..106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(121..126,127..144,145..151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    159..175
                     /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     164..186
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     183..199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     183..199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     218..238
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        220..238
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        266..492
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             493..673
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    720..845
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1338..1571
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1756..1800
                     /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        1978..2226
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1978..1998
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2149..2168
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2381..2498
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2567..2778
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2804..3403
                     /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(2804..2823)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3300..3320
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3419..4007
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3472..3492)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3890..3901)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4079..4312
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4390..4511
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4427..4446)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4481..4500
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4551..4686
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4706..4725)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4707..4725)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4735..4752)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4735..4751)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4744..4766)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4893..5348
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4980..4999)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5190..5211
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5374..5478
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5479..5547,5548..6339)
                     /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(5697..5716)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6510..7098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6999..7018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7252..7269
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
       61 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      121 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      181 cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga
      241 acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac
      301 atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga
      361 ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca
      421 tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct
      481 cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
      541 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
      601 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
      661 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
      721 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
      781 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
      841 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
      901 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
      961 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     1021 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     1081 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     1141 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
     1201 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     1261 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     1321 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     1381 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     1441 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     1501 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     1561 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     1621 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     1681 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     1741 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     1801 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     1861 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     1921 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccagagag
     1981 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     2041 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     2101 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     2161 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     2221 aacaccgaaa ccatcttata atcagcgttt aagagctatg ctggaaacag catagcaagt
     2281 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
     2341 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2401 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2461 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2521 gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca
     2581 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
     2641 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2701 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2761 tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatgaccg agtacaagcc
     2821 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2881 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     2941 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     3001 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3061 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3121 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3181 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3241 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3301 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3361 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     3421 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3481 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3541 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3601 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3661 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3721 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3781 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3841 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3901 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3961 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     4021 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4081 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4141 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4201 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4261 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     4321 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     4381 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4441 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4501 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     4561 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4621 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4681 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     4741 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4801 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4861 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     4921 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4981 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5041 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5101 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5161 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5221 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5281 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5341 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5401 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5461 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5521 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5581 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5641 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5701 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5761 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5821 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5881 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5941 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6001 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6061 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6121 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6181 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6241 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6301 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6361 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6421 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6481 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6541 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6601 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6661 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6721 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6781 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6841 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6901 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6961 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7021 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7081 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7141 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7201 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     7261 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc c
//
