LOCUS       sequence-183123-        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 F7.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..49
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    166..187
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(202..207,208..225,226..232)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    240..256
                     /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     245..267
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     264..280
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     264..280
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     299..319
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        301..319
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        347..573
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             574..754
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    801..926
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1419..1652
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1837..1881
                     /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        2059..2307
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2059..2079
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2230..2249
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2463..2580
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2649..2860
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2886..3485
                     /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(2886..2905)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3382..3402
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3501..4089
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3554..3574)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3972..3983)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4161..4394
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4472..4593
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4509..4528)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4563..4582
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4633..4768
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4788..4807)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4789..4807)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4817..4834)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4817..4833)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4826..4848)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4975..5430
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5062..5081)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5272..5293
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5456..5560
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5561..5629,5630..6421)
                     /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(5779..5798)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6592..7180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7081..7100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
       61 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      121 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      181 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      241 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
      301 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
      361 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
      421 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
      481 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
      541 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
      601 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      661 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      721 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      781 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      841 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      901 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      961 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     1021 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     1081 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1141 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1201 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1261 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     1321 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     1381 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     1441 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     1501 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     1561 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     1621 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     1681 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     1741 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     1801 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     1861 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     1921 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     1981 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     2041 accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga
     2101 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     2161 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     2221 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     2281 tatatcttgt ggaaaggacg aaacaccgtg aggagtgaat tgtaaggcgt ttaagagcta
     2341 tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg
     2401 caccgagtcg gtgctttttt tgaattctag atcttgagac aaatggcagt attcatccac
     2461 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     2521 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     2581 cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagtg gctccggtgc
     2641 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     2701 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2761 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2821 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg
     2881 ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     2941 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     3001 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     3061 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     3121 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     3181 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     3241 aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc
     3301 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     3361 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     3421 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     3481 cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
     3541 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3601 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     3661 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     3721 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     3781 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     3841 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     3901 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     3961 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     4021 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     4081 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     4141 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     4201 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     4261 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     4321 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     4381 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
     4441 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     4501 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4561 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     4621 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4681 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4741 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     4801 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     4861 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     4921 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     4981 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5041 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5101 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5161 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     5221 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5281 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5341 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5401 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     5461 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5521 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5581 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5641 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5701 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5761 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5821 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5881 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5941 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6001 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6061 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6121 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6181 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6241 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6301 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6361 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6421 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6481 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6541 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6601 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6661 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6721 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6781 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6841 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6901 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6961 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7021 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7081 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7141 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7201 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7261 tgattctgtg gataaccgta ttaccgcctt tgagtgagct ga
//
