LOCUS       sequence-183300-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDC34.
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: CDC34 B7.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    91..112
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(127..132,133..150,151..157)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    165..181
                     /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     170..192
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     189..205
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     189..205
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     224..244
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        226..244
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        272..498
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             499..679
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    726..851
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1344..1577
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1762..1806
                     /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        1984..2232
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1984..2004
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2155..2174
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2388..2505
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2574..2785
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2811..3410
                     /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(2811..2830)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3307..3327
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3426..4014
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3479..3499)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3897..3908)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4086..4319
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4397..4518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4434..4453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4488..4507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4558..4693
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4713..4732)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4714..4732)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4742..4759)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4742..4758)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4751..4773)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4900..5355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4987..5006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5197..5218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5381..5485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5486..5554,5555..6346)
                     /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(5704..5723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6517..7105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7006..7025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7259..7276
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
       61 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
      121 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      181 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      241 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
      301 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
      361 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
      421 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
      481 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      541 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
      601 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
      661 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
      721 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
      781 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
      841 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
      901 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
      961 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     1021 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     1081 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     1141 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     1201 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1261 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1321 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1381 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1441 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1501 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1561 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1621 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     1681 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     1741 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     1801 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     1861 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     1921 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     1981 agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2041 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2101 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2161 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     2221 ggacgaaaca ccggtcacgc ggaatccctc gacgtttaag agctatgctg gaaacagcat
     2281 agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     2341 ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     2401 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     2461 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2521 gacagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga
     2581 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     2641 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     2701 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     2761 caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc atgaccgagt
     2821 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     2881 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     2941 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3001 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3061 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3121 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3181 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3241 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3301 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3361 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag
     3421 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     3481 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3541 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3601 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3661 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3721 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3781 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     3841 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3901 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3961 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac
     4021 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     4081 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     4141 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     4201 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     4261 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     4321 tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
     4381 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4441 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4501 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc
     4561 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4621 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4681 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     4741 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4801 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4861 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     4921 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4981 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     5041 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     5101 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5161 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5221 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     5281 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5341 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5581 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5641 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5701 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5761 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5821 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5881 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5941 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6001 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6061 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6121 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6181 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6241 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6301 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6361 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6661 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7141 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7201 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7261 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aa
//
