LOCUS       sequence-183169-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human DYNLT1.
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: DYNLT1 B2.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..95
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    212..233
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(248..253,254..271,272..278)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    286..302
                     /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     291..313
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     310..326
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     310..326
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     345..365
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        347..365
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        393..619
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             620..800
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    847..972
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1465..1698
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1883..1927
                     /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        2105..2353
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2105..2125
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2276..2295
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2509..2626
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2695..2906
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2932..3531
                     /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(2932..2951)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3428..3448
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3547..4135
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3600..3620)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4018..4029)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4207..4440
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4518..4639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4555..4574)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4609..4628
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4679..4814
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4834..4853)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4835..4853)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4863..4880)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4863..4879)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4872..4894)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5021..5476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5108..5127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5318..5339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5502..5606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5607..5675,5676..6467)
                     /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(5825..5844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6638..7226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7127..7146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
       61 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
      121 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
      181 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      241 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      301 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
      361 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
      421 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
      481 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
      541 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
      601 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
      661 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
      721 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
      781 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
      841 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
      901 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
      961 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     1021 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     1081 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     1141 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     1201 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     1261 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     1321 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1381 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1441 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1501 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1561 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1621 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     1681 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     1741 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     1801 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     1861 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     1921 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     1981 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     2041 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     2101 cagagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2161 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2221 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2281 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2341 aggacgaaac accgctacta catttgtggt ccacgtttaa gagctatgct ggaaacagca
     2401 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2461 tttttttgaa ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2521 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2581 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2641 ggacagcaga gatccacttt ggcgccggct cgagtggctc cggtgcccgt cagtgggcag
     2701 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     2761 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     2821 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     2881 gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac catgaccgag
     2941 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     3001 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     3061 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3121 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3181 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3241 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3301 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3361 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3421 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     3481 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     3541 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3601 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3661 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3721 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3781 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3841 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3901 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3961 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4021 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4081 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     4141 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4201 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4261 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4321 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4381 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4441 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     4501 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4561 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4621 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     4681 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4741 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4801 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     4861 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4921 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4981 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     5041 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5101 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5161 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5221 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5281 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5341 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5401 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5461 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5521 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5581 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5641 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5701 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5761 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5821 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5881 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5941 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6001 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6061 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6301 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6781 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7261 ccttttgctg gccttttgct cacatgttct ttcctgcgtt at
//
