LOCUS       sequence-183039-        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 F1.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..41
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    158..179
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(194..199,200..217,218..224)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    232..248
                     /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     237..259
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     256..272
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     256..272
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     291..311
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        293..311
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        339..565
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             566..746
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    793..918
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1411..1644
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1829..1873
                     /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        2051..2299
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2051..2071
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2222..2241
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2641..2852
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2878..3477
                     /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(2878..2897)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3374..3394
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3493..4081
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3546..3566)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3964..3975)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4153..4386
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4464..4585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4501..4520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4555..4574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4625..4760
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4780..4799)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4781..4799)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4809..4826)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4809..4825)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4818..4840)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4967..5422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5054..5073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5264..5285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5448..5552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5553..5621,5622..6413)
                     /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(5771..5790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6584..7172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7073..7092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
       61 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
      121 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
      181 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      241 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
      301 ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt
      361 gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca
      421 ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca
      481 gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt
      541 taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga
      601 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
      661 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
      721 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
      781 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
      841 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
      901 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
      961 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     1021 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     1081 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     1141 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     1201 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     1261 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     1321 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     1381 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     1441 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     1501 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     1561 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     1621 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     1681 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     1741 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     1801 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     1861 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     1921 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     1981 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     2041 gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     2101 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     2161 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     2221 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     2281 gtggaaagga cgaaacaccg ctgacccact cactgggcgg gtttaagagc tatgctggaa
     2341 acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     2401 cggtgctttt tttgaattct agatcttgag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt gcccgtcagt
     2641 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     2701 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     2761 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     2821 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc cgccaccatg
     2881 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     2941 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     3001 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     3061 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     3121 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     3181 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     3241 gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg tctgggcagc
     3301 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     3361 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     3421 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg
     3481 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3541 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3601 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3661 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3721 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3781 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3841 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     3901 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3961 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4021 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4081 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     4141 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     4201 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4261 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4321 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     4381 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4441 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     4501 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4561 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     4621 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4681 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4741 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     4801 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4861 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4921 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4981 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5041 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5101 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5161 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5221 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5281 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5341 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5401 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5461 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5521 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5581 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5641 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5701 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5761 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5821 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5881 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5941 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6001 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6061 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6121 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6181 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6241 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6301 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6361 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6421 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6481 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6541 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6601 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6661 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6721 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6781 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6841 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6901 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6961 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7021 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7081 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7141 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7201 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7261 tggataaccg tattaccgcc tttgagtgag ctgataccgc tc
//
