LOCUS       sequence-183131-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human UBE2C.
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: UBE2C H6.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..19
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    136..157
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(172..177,178..195,196..202)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    210..226
                     /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     215..237
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     234..250
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     234..250
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     269..289
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        271..289
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        317..543
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             544..724
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    771..896
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1389..1622
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1807..1851
                     /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        2029..2277
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2029..2049
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2200..2219
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2432..2549
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2618..2829
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2855..3454
                     /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(2855..2874)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3351..3371
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3470..4058
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3523..3543)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3941..3952)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4130..4363
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4441..4562
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4478..4497)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4532..4551
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4602..4737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4757..4776)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4758..4776)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4786..4803)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4786..4802)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4795..4817)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4944..5399
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5031..5050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5241..5262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5425..5529
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5530..5598,5599..6390)
                     /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(5748..5767)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6561..7149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7050..7069
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
       61 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
      121 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
      181 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      241 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
      301 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
      361 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
      421 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
      481 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
      541 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      601 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      661 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
      721 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
      781 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
      841 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
      901 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
      961 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1021 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1081 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1141 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1201 agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga
     1261 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1321 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1381 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1441 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1501 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1561 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1621 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1681 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1741 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     1801 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     1861 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     1921 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     1981 tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga gggcctattt
     2041 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
     2101 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     2161 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     2221 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgcc
     2281 ctgctatcac cccaacggtt taagagctat gctggaaaca gcatagcaag tttaaataag
     2341 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattctaga
     2401 tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     2461 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     2521 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     2581 tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     2641 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     2701 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2761 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     2821 agaacacagg tgtcgtgacg cgggatccgc caccatgacc gagtacaagc ccacggtgcg
     2881 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     2941 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     3001 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     3061 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     3121 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     3181 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     3241 agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     3301 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     3361 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     3421 cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct
     3481 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3541 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3601 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     3661 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     3721 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     3781 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3841 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     3901 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3961 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4021 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact
     4081 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     4141 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     4201 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     4261 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     4321 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca
     4381 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     4441 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4501 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4561 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     4621 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4681 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4741 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     4801 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4861 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4921 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4981 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5041 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5101 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5161 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5221 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5281 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5341 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5401 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5461 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5521 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5581 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5641 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5701 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5761 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5821 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5881 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5941 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6001 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6061 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6121 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6181 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6241 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6301 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6361 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6421 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6481 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6541 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6601 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6661 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6721 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6781 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6841 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6901 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6961 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7021 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7081 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7141 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7201 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7261 gagtgagctg ataccgctcg ccgcagccga acgaccgagc g
//
