LOCUS       sequence-183181-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ELAVL1.
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: ELAVL1 B11.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..42
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    159..180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(195..200,201..218,219..225)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    233..249
                     /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     238..260
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     257..273
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     257..273
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     292..312
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        294..312
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        340..566
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             567..747
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    794..919
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1412..1645
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1830..1874
                     /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        2052..2300
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2052..2072
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2223..2242
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2456..2573
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2642..2853
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2879..3478
                     /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(2879..2898)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3375..3395
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3494..4082
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3547..3567)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3965..3976)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4154..4387
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4465..4586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4502..4521)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4556..4575
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4626..4761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4781..4800)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4782..4800)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4810..4827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4810..4826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4819..4841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4968..5423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5055..5074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5265..5286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5449..5553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5554..5622,5623..6414)
                     /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(5772..5791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6585..7173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7074..7093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
       61 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      121 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      181 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      241 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      301 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
      361 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
      421 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
      481 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
      541 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
      601 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
      661 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
      721 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
      781 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
      841 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
      901 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
      961 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     1021 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     1081 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     1141 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1201 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1261 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     1321 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1381 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1441 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1501 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1561 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1621 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     1681 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     1741 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     1801 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     1861 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     1921 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     1981 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     2041 ggggacccag agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2101 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2161 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2221 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2281 tgtggaaagg acgaaacacc gtccttctgg gtcatggtcc ggtttaagag ctatgctgga
     2341 aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     2401 tcggtgcttt ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta
     2461 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     2521 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     2581 attacaggga cagcagagat ccactttggc gccggctcga gtggctccgg tgcccgtcag
     2641 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2701 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2761 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2821 ctttttcgca acgggtttgc cgccagaaca caggtgtcgt gacgcgggat ccgccaccat
     2881 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     2941 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     3001 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     3061 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     3121 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3181 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3241 cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag
     3301 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3361 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3421 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
     3481 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3541 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3601 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     3661 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     3721 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     3781 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     3841 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3901 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     3961 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     4021 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     4081 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     4141 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     4201 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     4261 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     4321 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     4381 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     4441 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     4501 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4561 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     4621 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4681 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4741 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     4801 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4861 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4921 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     4981 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5041 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5101 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5161 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5221 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5281 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5341 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5401 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5461 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5521 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5581 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5641 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5701 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5761 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5821 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5881 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5941 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6001 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6061 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6121 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6181 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6241 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6301 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6361 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6421 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6481 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6541 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6601 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6661 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6721 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6781 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6841 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6901 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6961 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7021 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7081 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7141 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7201 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7261 gtggataacc gtattaccgc ctttgagtga gctgataccg ct
//
