LOCUS       sequence-190325-        7327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human DYNLT3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7327)
  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 7327)
  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: DYNLT3 B3.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..70
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(85..90,91..108,109..115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    123..139
                     /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     128..150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     147..163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     147..163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     182..202
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        184..202
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        230..456
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             457..637
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    684..809
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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        1942..2190
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1942..1962
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2113..2132
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2371..2488
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2557..2768
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2794..3393
                     /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(2794..2813)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3290..3310
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3409..3997
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3462..3482)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3880..3891)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4069..4302
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4380..4501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4417..4436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4471..4490
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4541..4676
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4696..4715)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4697..4715)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4725..4742)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4725..4741)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4734..4756)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4883..5338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4970..4989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5180..5201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5364..5468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5469..5537,5538..6329)
                     /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(5687..5706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6500..7088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6989..7008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7242..7259
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
       61 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      121 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      181 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      241 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      301 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      361 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      421 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      481 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      541 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      601 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      661 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      721 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      781 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      841 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      901 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      961 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1021 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1081 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1141 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     1981 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2041 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2101 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2161 ttatatatct tgtggaaagg acgaaacacc gaggcttttc ccaacttaac cgtaagagct
     2221 atgctggaaa cagtaaccgt aagagctatg ctggaaacag catagcaagt ttaaataagg
     2281 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg aattctagat
     2341 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2401 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2461 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
     2521 ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     2581 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     2641 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     2701 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     2761 gaacacaggt gtcgtgacgc gggatccgcc accatgaccg agtacaagcc cacggtgcgc
     2821 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     2881 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     2941 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3001 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     3061 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     3121 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     3181 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     3241 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     3301 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     3361 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg
     3421 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3481 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3541 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3601 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3661 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3721 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3781 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     3841 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3901 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     3961 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     4021 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     4081 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     4141 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4201 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4261 gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat
     4321 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     4381 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4441 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4501 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     4561 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4621 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4681 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     4741 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4801 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4861 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4921 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4981 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5041 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5101 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5161 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5221 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5281 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     5341 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5401 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5461 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5521 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5581 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5641 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5701 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5761 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5821 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5881 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5941 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6001 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6061 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6181 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     7261 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     7321 gcagctg
//
