LOCUS       sequence-183227-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human FBXO5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  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 7301)
  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: FBXO5 D4.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..57
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    174..195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(210..215,216..233,234..240)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    248..264
                     /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     253..275
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     272..288
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     272..288
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     307..327
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        309..327
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        355..581
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             582..762
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    809..934
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1427..1660
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1845..1889
                     /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        2067..2315
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2067..2087
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2238..2257
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2470..2587
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2656..2867
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2893..3492
                     /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(2893..2912)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3389..3409
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3508..4096
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3561..3581)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3979..3990)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4168..4401
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4479..4600
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4516..4535)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4570..4589
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4640..4775
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4795..4814)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4796..4814)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4824..4841)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4824..4840)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4833..4855)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4982..5437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5069..5088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5279..5300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5463..5567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5568..5636,5637..6428)
                     /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(5786..5805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6599..7187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7088..7107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
       61 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      121 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      181 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      241 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      301 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
      361 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
      421 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
      481 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
      541 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
      601 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      661 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      721 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      781 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      841 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      901 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      961 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     1021 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     1081 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1141 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1201 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1261 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1321 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1381 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1441 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1501 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1561 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1621 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1681 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1741 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1801 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1861 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1921 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1981 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     2041 acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc
     2101 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2161 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2221 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2281 tggctttata tatcttgtgg aaaggacgaa acaccgttta agtccggaat gaacagttta
     2341 agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa
     2401 aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa tggcagtatt
     2461 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2521 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2581 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     2641 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2701 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2761 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2821 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     2881 ggatccgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     2941 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     3001 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     3061 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     3121 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     3181 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3241 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     3301 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3361 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3421 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3481 cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga
     3541 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3601 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3661 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3721 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3781 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3841 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3901 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3961 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     4021 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     4081 tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta
     4141 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     4201 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     4261 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     4321 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     4381 cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt
     4441 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata
     4501 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4561 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat
     4621 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4681 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4741 attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat
     4801 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4861 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     4921 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     4981 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5041 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5101 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5161 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5221 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5281 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5341 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5401 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     5461 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5521 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5581 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5641 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5701 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5761 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5821 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5881 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5941 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6001 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6061 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6121 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6181 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6241 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6301 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6361 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6421 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6481 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6541 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6601 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6661 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6721 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6781 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6841 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6901 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6961 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7021 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7081 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7141 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7201 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7261 tatcccctga ttctgtggat aaccgtatta ccgcctttga g
//
