LOCUS       sequence-183323-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human COPS3.
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: COPS3 G12.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    108..129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(144..149,150..167,168..174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    182..198
                     /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     187..209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     206..222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     206..222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     241..261
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        243..261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        289..515
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             516..696
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    743..868
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1361..1594
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1779..1823
                     /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        2001..2249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2001..2021
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2172..2191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2405..2522
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2591..2802
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2828..3427
                     /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(2828..2847)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3324..3344
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3443..4031
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3496..3516)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3914..3925)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4103..4336
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4414..4535
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4451..4470)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4505..4524
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4575..4710
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4730..4749)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4731..4749)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4759..4776)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4759..4775)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4768..4790)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4917..5372
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5004..5023)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5214..5235
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5398..5502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5503..5571,5572..6363)
                     /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(5721..5740)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6534..7122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7023..7042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7276..7293
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
       61 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      121 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      181 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
      241 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
      301 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
      361 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
      421 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
      481 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
      541 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      601 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      661 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      721 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      781 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
      841 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
      901 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
      961 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1021 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1081 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1141 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1201 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     1261 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     1321 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     1381 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     1441 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     1501 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     1561 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     1621 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     1681 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     1741 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     1801 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     1861 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     1921 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     1981 caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac
     2041 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     2101 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2161 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2221 tatatatctt gtggaaagga cgaaacaccg gtgaactgat caacaagagt gtttaagagc
     2281 tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
     2341 ggcaccgagt cggtgctttt tttgaattct agatcttgag acaaatggca gtattcatcc
     2401 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2461 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2521 ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt
     2581 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2641 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2701 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2761 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc
     2821 cgccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     2881 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     2941 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3001 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3061 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3121 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3181 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     3241 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3301 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3361 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3421 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     3481 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3541 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3601 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     3661 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     3721 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     3781 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     3841 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     3901 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     3961 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     4021 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     4081 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     4141 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     4201 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     4261 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     4321 tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa
     4381 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt
     4441 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4501 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc
     4561 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4621 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4681 agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga
     4741 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     4801 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     4861 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     4921 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4981 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5041 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5101 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5161 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5221 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5281 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5341 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     5401 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5461 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5521 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5581 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5641 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5701 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5761 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5821 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5881 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5941 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6001 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6061 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6121 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6181 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6241 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6301 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6361 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6421 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6481 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6541 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6601 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6661 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6721 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6781 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6841 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6901 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6961 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7021 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7081 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7141 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7201 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7261 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aa
//
