LOCUS       sequence-183341-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human DCTN5.
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: DCTN5 C1.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..40
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    157..178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(193..198,199..216,217..223)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    231..247
                     /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     236..258
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     255..271
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     255..271
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     290..310
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        292..310
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        338..564
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             565..745
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    792..917
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1410..1643
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1828..1872
                     /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        2050..2298
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2050..2070
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2221..2240
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2454..2571
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2640..2851
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2877..3476
                     /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(2877..2896)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3373..3393
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3492..4080
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3545..3565)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3963..3974)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4152..4385
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4463..4584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4500..4519)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4554..4573
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4624..4759
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4779..4798)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4780..4798)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4808..4825)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4808..4824)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4817..4839)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4966..5421
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5053..5072)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5263..5284
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5447..5551
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5552..5620,5621..6412)
                     /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(5770..5789)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6583..7171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7072..7091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
       61 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      121 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      181 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      241 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
      301 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
      361 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
      421 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
      481 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
      541 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
      601 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
      661 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
      721 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
      781 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
      841 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
      901 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
      961 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1021 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1081 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1141 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1201 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1261 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1321 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1381 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1441 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1501 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1561 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1621 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1681 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1741 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1801 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1861 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1921 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1981 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2041 ggacccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2101 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2161 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2221 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2281 tggaaaggac gaaacaccgt gccattgaga acgatgttcg tttaagagct atgctggaaa
     2341 cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     2401 ggtgcttttt ttgaattcta gatcttgaga caaatggcag tattcatcca caattttaaa
     2461 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     2521 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     2581 tacagggaca gcagagatcc actttggcgc cggctcgagt ggctccggtg cccgtcagtg
     2641 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
     2701 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     2761 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     2821 ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc gccaccatga
     2881 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     2941 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     3001 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3061 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3121 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3181 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3241 cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3301 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3361 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3421 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc
     3481 gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3541 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3601 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3661 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3721 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3781 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3841 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3901 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3961 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     4021 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4081 gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     4141 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
     4201 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     4261 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     4321 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     4381 tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa
     4441 atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag
     4501 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4561 gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg
     4621 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4681 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4741 ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg
     4801 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4861 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4921 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     4981 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5041 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5101 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5161 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     5221 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5281 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     5341 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5401 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5461 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5521 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5581 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5641 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5701 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5761 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5821 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5881 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5941 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6001 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6061 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6121 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6181 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6241 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6301 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6361 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6421 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6481 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6541 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6601 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6661 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6721 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6781 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6841 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6901 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6961 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7021 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7081 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7141 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7201 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     7261 ggataaccgt attaccgcct ttgagtgagc tgataccgct cg
//
