LOCUS       sequence-183037-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TUBB.
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: TUBB F2.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    128..149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(164..169,170..187,188..194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    202..218
                     /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     207..229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     226..242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     226..242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     261..281
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        263..281
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        309..535
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             536..716
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    763..888
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1381..1614
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1799..1843
                     /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        2021..2269
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2021..2041
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2192..2211
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2425..2542
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2611..2822
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2848..3447
                     /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(2848..2867)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3344..3364
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3463..4051
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3516..3536)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3934..3945)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4123..4356
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4434..4555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4471..4490)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4525..4544
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4595..4730
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4750..4769)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4751..4769)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4779..4796)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4779..4795)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4788..4810)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4937..5392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5024..5043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5234..5255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5418..5522
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5523..5591,5592..6383)
                     /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(5741..5760)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6554..7142
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7043..7062
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7296..7302,1..11)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
       61 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      121 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      181 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      241 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
      301 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
      361 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      421 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      481 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      721 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      781 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      841 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      901 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      961 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1021 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1081 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1141 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1201 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1261 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1321 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1381 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1441 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1501 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1561 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1621 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1681 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1741 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1801 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1861 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1921 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1981 atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat
     2041 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     2101 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2161 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2221 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg aaggtggttc
     2281 agatccccgt gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc
     2341 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgaattct agatcttgag
     2401 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     2461 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     2521 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg
     2581 ccggctcgag tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     2641 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     2701 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     2761 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     2821 aggtgtcgtg acgcgggatc cgccaccatg accgagtaca agcccacggt gcgcctcgcc
     2881 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     2941 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3001 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3061 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3121 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3181 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
     3241 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3301 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     3361 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     3421 tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac
     3481 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3541 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3601 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3661 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3721 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3781 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3841 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     3901 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3961 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     4021 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
     4081 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4141 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     4201 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4261 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4321 tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc
     4381 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4441 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4501 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4561 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4621 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4681 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     4741 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     4801 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4861 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4921 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5341 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5401 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5461 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5521 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5581 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5641 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5701 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5761 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5821 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5881 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5941 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6001 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6061 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6121 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6181 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6241 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6301 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6361 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6421 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6481 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6541 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6601 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6661 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6721 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6781 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6841 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6901 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6961 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7021 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7081 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7141 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7201 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7261 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gt
//
