LOCUS       sequence-183125-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TUBG1.
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: TUBG1 F8.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..66
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    183..204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(219..224,225..242,243..249)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    257..273
                     /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     262..284
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     281..297
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     281..297
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     316..336
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        318..336
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        364..590
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             591..771
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    818..943
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1436..1669
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1854..1898
                     /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        2076..2324
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2076..2096
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2247..2266
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2480..2597
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2666..2877
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2903..3502
                     /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(2903..2922)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3399..3419
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3518..4106
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3571..3591)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3989..4000)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4178..4411
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4489..4610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4526..4545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4580..4599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4650..4785
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4805..4824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4806..4824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4834..4851)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4834..4850)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4843..4865)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4992..5447
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5079..5098)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5289..5310
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5473..5577
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5578..5646,5647..6438)
                     /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(5796..5815)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6609..7197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7098..7117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
       61 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      121 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      181 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      241 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      301 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
      361 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
      421 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
      481 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
      541 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
      601 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
      661 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
      721 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
      781 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
      841 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      901 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      961 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     1021 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     1081 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     1141 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     1201 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     1261 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     1321 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1381 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1441 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1501 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1561 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1621 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1681 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1741 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     1801 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     1861 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     1921 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     1981 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     2041 ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt
     2101 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     2161 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     2221 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2281 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgtca ttcagccgtt
     2341 ctaaggttta agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta
     2401 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa
     2461 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     2521 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2581 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc
     2641 tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     2701 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2761 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2821 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg
     2881 tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     2941 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     3001 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3061 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3121 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3181 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3241 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     3301 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     3361 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     3421 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     3481 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     3541 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3601 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3661 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3721 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3781 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3841 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3901 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     3961 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     4021 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     4081 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     4141 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     4201 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     4261 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     4321 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     4381 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     4441 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4501 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4561 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4621 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4681 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4741 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4801 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4861 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4921 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4981 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5041 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5101 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5161 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5221 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5281 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5341 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5401 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5461 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5521 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5581 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5641 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5701 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5761 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5821 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5881 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5941 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6001 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6061 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6121 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6181 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6241 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6301 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6361 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6421 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6481 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6541 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6601 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6661 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6721 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6781 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6841 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6901 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6961 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7021 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7081 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7141 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7201 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7261 tttcctgcgt tatcccctga ttctgtggat aaccgtatta cc
//
