LOCUS       sequence-183364-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human RRM2.
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: RRM2 E4.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..23
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    140..161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(176..181,182..199,200..206)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    214..230
                     /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     219..241
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     238..254
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     238..254
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     273..293
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        275..293
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        321..547
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             548..728
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    775..900
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1393..1626
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1811..1855
                     /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        2033..2281
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2033..2053
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2204..2223
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2437..2554
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2623..2834
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2860..3459
                     /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(2860..2879)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3356..3376
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3475..4063
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3528..3548)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3946..3957)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4135..4368
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4446..4567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4483..4502)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4537..4556
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4607..4742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4762..4781)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4763..4781)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4791..4808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4791..4807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4800..4822)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4949..5404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5036..5055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5246..5267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5430..5534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5535..5603,5604..6395)
                     /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(5753..5772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6566..7154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7055..7074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
       61 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
      121 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
      181 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
      241 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
      301 agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
      361 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
      421 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
      481 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
      541 cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
      601 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
      661 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
      721 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
      781 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
      841 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
      901 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
      961 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     1021 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     1081 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     1141 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     1201 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     1261 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     1321 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     1381 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     1441 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     1501 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     1561 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     1621 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     1681 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     1741 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     1801 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     1861 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     1921 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     1981 atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca gagagggcct
     2041 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
     2101 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     2161 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     2221 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     2281 cgccggccct tcccattggc ttgtttaaga gctatgctgg aaacagcata gcaagtttaa
     2341 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt
     2401 ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     2461 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     2521 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     2581 tccactttgg cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc
     2641 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
     2701 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2761 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2821 ccgccagaac acaggtgtcg tgacgcggga tccgccacca tgaccgagta caagcccacg
     2881 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     2941 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     3001 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3061 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3121 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3181 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3241 gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3301 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3361 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3421 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa
     3481 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3541 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3601 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3661 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3721 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3781 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3841 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     3901 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3961 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     4021 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     4081 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     4141 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     4201 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     4261 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     4321 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     4381 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     4441 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4501 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4561 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     4621 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4681 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4741 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     4801 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4861 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4921 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4981 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5041 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5101 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5161 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5221 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5281 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5341 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5401 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5461 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5521 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5581 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5641 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5701 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5761 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5821 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5881 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5941 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6001 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6061 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6121 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6181 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6241 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6301 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6361 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6421 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6481 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6541 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6601 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6661 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6721 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6781 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6841 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6901 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6961 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7021 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7081 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7141 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7201 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7261 cctttgagtg agctgatacc gctcgccgca gccgaacgac cg
//
