LOCUS       sequence-183145-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TUBE1.
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: TUBE1 F6.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..111
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    228..249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(264..269,270..287,288..294)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    302..318
                     /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     307..329
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     326..342
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     326..342
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     361..381
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        363..381
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        409..635
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             636..816
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    863..988
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1481..1714
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1899..1943
                     /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        2121..2369
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2121..2141
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2292..2311
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2525..2642
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2711..2922
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2948..3547
                     /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(2948..2967)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3444..3464
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3563..4151
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3616..3636)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4034..4045)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4223..4456
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4534..4655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4571..4590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4625..4644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4695..4830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4850..4869)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4851..4869)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4879..4896)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4879..4895)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4888..4910)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5037..5492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5124..5143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5334..5355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5518..5622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5623..5691,5692..6483)
                     /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(5841..5860)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6654..7242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7143..7162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      301 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
      361 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
      421 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
      481 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
      541 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
      601 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
      661 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      721 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      781 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      841 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      901 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
      961 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1021 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1081 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1141 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1201 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1261 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1321 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     1381 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     1441 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     1501 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     1561 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     1621 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     1681 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     1741 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     1801 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     1861 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     1921 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     1981 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     2041 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     2101 caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac
     2161 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     2221 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2281 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2341 tatatatctt gtggaaagga cgaaacaccg atcctcacca gaaggataaa gtttaagagc
     2401 tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
     2461 ggcaccgagt cggtgctttt tttgaattct agatcttgag acaaatggca gtattcatcc
     2521 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2581 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2641 ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt
     2701 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2761 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2821 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2881 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc
     2941 cgccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     3001 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     3061 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3121 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3181 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     3241 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     3301 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     3361 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3421 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3481 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3541 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     3601 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3661 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3721 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     3781 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     3841 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     3901 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     3961 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     4021 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     4081 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     4141 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     4201 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     4261 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     4321 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     4381 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     4441 tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa
     4501 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt
     4561 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4621 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc
     4681 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4741 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4801 agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga
     4861 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     4921 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     4981 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     5041 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5101 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5161 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5221 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5281 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5341 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5401 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5461 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     5521 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5581 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5641 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5701 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5761 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5821 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5881 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5941 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6001 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6061 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6121 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6181 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6241 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6301 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6361 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6421 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6481 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7261 tttttacggt tcctggcctt ttgctggcct tttgctcaca tg
//
