LOCUS       sequence-183136-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TUBD1.
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: TUBD1 F4.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..26
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    143..164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(179..184,185..202,203..209)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    217..233
                     /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     222..244
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     241..257
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     241..257
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     276..296
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        278..296
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        324..550
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             551..731
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    778..903
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1396..1629
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1814..1858
                     /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        2036..2284
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2036..2056
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2207..2226
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2626..2837
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2863..3462
                     /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(2863..2882)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3359..3379
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3478..4066
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3531..3551)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3949..3960)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4138..4371
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4449..4570
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4486..4505)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4540..4559
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4610..4745
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4765..4784)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4766..4784)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4794..4811)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4794..4810)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4803..4825)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4952..5407
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5039..5058)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5249..5270
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5433..5537
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5538..5606,5607..6398)
                     /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(5756..5775)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6569..7157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7058..7077
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
       61 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      121 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      181 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      301 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
      361 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
      421 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
      481 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
      541 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      601 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      661 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      721 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      781 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
      841 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
      901 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
      961 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     1021 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     1081 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     1141 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     1201 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     1261 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     1321 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     1381 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     1441 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     1501 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     1561 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     1621 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     1681 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     1741 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     1801 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     1861 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     1921 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     1981 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg
     2041 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2101 tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2161 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2221 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2281 caccgatatt tccattggcc agactgttta agagctatgc tggaaacagc atagcaagtt
     2341 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga
     2401 attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca gagcgcacat
     2641 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
     2701 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2761 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2821 ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc
     2881 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     2941 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     3001 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     3061 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     3121 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     3181 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     3241 accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     3301 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     3361 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     3421 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat
     3481 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3541 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3601 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3661 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3721 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3781 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3841 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3901 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3961 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     4021 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     4081 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     4141 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     4201 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     4261 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     4321 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
     4381 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     4441 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4501 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4561 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     4621 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4681 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4741 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     4801 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4861 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4921 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4981 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5041 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5101 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5161 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5221 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5281 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5341 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5401 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5461 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5521 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5581 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5641 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5701 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5761 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5821 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5881 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5941 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6001 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6061 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6121 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6181 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6241 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6301 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6361 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6421 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6481 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6541 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6601 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6661 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6721 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6781 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6841 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6901 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6961 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7021 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7081 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7141 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7201 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7261 ccgcctttga gtgagctgat accgctcgcc gcagccgaac ga
//
