LOCUS       sequence-183925-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human RFC4.
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: RFC4 D6.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    133..154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(169..174,175..192,193..199)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    207..223
                     /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     212..234
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     231..247
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     231..247
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     266..286
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        268..286
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        314..540
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             541..721
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    768..893
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1386..1619
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1804..1848
                     /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        2026..2274
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2026..2046
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2197..2216
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2430..2547
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2616..2827
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2853..3452
                     /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(2853..2872)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3349..3369
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3468..4056
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3521..3541)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3939..3950)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4128..4361
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4439..4560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4476..4495)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4530..4549
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4600..4735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4755..4774)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4756..4774)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4784..4801)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4784..4800)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4793..4815)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4942..5397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5029..5048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5239..5260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5423..5527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5528..5596,5597..6388)
                     /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(5746..5765)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6559..7147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7048..7067
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7301..7302,1..16)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
       61 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      121 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      181 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      241 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
      301 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg
      361 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta
      421 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga
      481 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac
      541 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
      601 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      661 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      721 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      781 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      841 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      901 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      961 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
     1021 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
     1081 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
     1141 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
     1201 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
     1261 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1321 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1381 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1441 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1501 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1561 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1621 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1681 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1741 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1801 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1861 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1921 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1981 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc
     2041 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     2101 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     2161 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     2221 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcagcg
     2281 ggggtttagt actgagttta agagctatgc tggaaacagc atagcaagtt taaataaggc
     2341 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attctagatc
     2401 ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     2461 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     2521 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt
     2581 tggcgccggc tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2641 ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
     2701 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2761 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     2821 aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc acggtgcgcc
     2881 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     2941 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     3001 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     3061 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     3121 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     3181 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag
     3241 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     3301 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     3361 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     3421 cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg
     3481 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3541 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3601 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3661 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3721 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3781 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3841 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     3901 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3961 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     4021 agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta
     4081 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     4141 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     4201 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     4261 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     4321 atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg
     4381 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa
     4441 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4501 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4561 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
     4621 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4681 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg
     4741 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     4801 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4861 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4921 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4981 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5041 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5101 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5161 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5221 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5281 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5341 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     5401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5461 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5521 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5581 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5641 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5701 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5761 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5821 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5881 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5941 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6001 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6061 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6121 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6181 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6241 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6301 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6361 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6421 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6481 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6541 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6601 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6661 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6721 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6781 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6841 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6901 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6961 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7021 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7081 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7141 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7201 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     7261 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc ag
//
