LOCUS       sequence-183298-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDC45.
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: CDC45 B9.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..80
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    197..218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(233..238,239..256,257..263)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    271..287
                     /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     276..298
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     295..311
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     295..311
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     330..350
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        332..350
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        378..604
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             605..785
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    832..957
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1450..1683
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1868..1912
                     /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        2090..2338
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2090..2110
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2261..2280
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2493..2610
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2679..2890
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2916..3515
                     /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(2916..2935)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3412..3432
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3531..4119
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3584..3604)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4002..4013)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4191..4424
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4502..4623
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4539..4558)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4593..4612
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4663..4798
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4818..4837)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4819..4837)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4847..4864)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4847..4863)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4856..4878)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5005..5460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5092..5111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5302..5323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5486..5590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5591..5659,5660..6451)
                     /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(5809..5828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6622..7210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7111..7130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
       61 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
      121 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      181 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      241 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      301 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
      361 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
      421 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
      481 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
      541 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
      601 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      661 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      721 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      781 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
      841 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
      901 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
      961 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     1021 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     1081 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1141 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1201 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1261 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     1321 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     1381 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     1441 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     1501 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     1561 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     1621 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     1681 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     1741 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     1801 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     1861 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     1921 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     1981 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     2041 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     2101 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     2161 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2221 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2281 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     2341 accagcgtat attgcacggt ttaagagcta tgctggaaac agcatagcaa gtttaaataa
     2401 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattctag
     2461 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2521 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2581 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2641 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2701 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2761 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2821 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2881 cagaacacag gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag cccacggtgc
     2941 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     3001 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     3061 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     3121 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     3181 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     3241 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     3301 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     3361 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     3421 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     3481 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
     3541 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3601 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3661 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3721 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3781 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3841 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3901 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     3961 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     4021 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4081 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     4141 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     4201 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     4261 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4321 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4381 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc
     4441 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     4501 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4561 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4621 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     4681 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4741 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4801 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     4861 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4921 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4981 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5041 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5101 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5161 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5221 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5281 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5341 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5401 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5461 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5521 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5581 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5641 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5701 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5761 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5821 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5881 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5941 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6001 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6061 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6121 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6181 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6241 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6301 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6361 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6421 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6601 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6661 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6721 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6781 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6841 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6901 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6961 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7021 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7081 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7141 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7201 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7261 tgctcacatg ttctttcctg cgttatcccc tgattctgtg g
//
