LOCUS       sequence-183362-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human RNASEH2A.
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: RNASEH2A B10.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    132..153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(168..173,174..191,192..198)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    206..222
                     /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     211..233
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     230..246
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     230..246
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     265..285
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        267..285
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        313..539
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             540..720
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    767..892
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1385..1618
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1803..1847
                     /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        2025..2273
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2025..2045
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2196..2215
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2429..2546
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2615..2826
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2852..3451
                     /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(2852..2871)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3348..3368
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3467..4055
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3520..3540)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3938..3949)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4127..4360
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4438..4559
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4475..4494)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4529..4548
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4599..4734
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4754..4773)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4755..4773)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4783..4800)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4783..4799)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4792..4814)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4941..5396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5028..5047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5238..5259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5422..5526
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5527..5595,5596..6387)
                     /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(5745..5764)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6558..7146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7047..7066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7300..7302,1..15)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
       61 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
      121 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
      181 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
      241 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
      301 ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga
      361 gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
      421 ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
      481 ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg
      541 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      601 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
      661 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
      721 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
      781 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      841 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      901 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      961 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     1021 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     1081 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     1141 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     1201 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
     1261 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1321 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1381 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1441 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1501 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1561 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     1621 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     1681 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     1741 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     1801 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     1861 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     1921 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     1981 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cagagagggc ctatttccca
     2041 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     2101 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     2161 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     2221 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgccgcgg
     2281 gcacaggcga actcgtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct
     2341 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa ttctagatct
     2401 tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     2461 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     2521 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt
     2581 ggcgccggct cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2641 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     2701 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2761 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2821 acacaggtgt cgtgacgcgg gatccgccac catgaccgag tacaagccca cggtgcgcct
     2881 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     2941 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     3001 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     3061 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     3121 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     3181 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     3241 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     3301 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     3361 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     3421 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
     3481 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3541 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3601 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3661 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3721 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3781 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3841 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     3901 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3961 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4021 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     4081 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     4141 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     4201 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     4261 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     4321 tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt
     4381 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     4441 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4501 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4561 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4621 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4681 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     4741 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     4801 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     4861 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     4921 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4981 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5041 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5101 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     5161 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5221 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5281 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5341 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     5401 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5461 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5521 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5581 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5641 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5701 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5761 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5821 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5881 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5941 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6001 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6061 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6121 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6181 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6241 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6301 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6361 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6421 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6481 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6541 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6601 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6661 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6721 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6781 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6841 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6901 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6961 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7021 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7081 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7141 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7201 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7261 tgagctgata ccgctcgccg cagccgaacg accgagcgca gc
//
