LOCUS       sequence-183148-        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 F5.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..36
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(51..56,57..74,75..81)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    89..105
                     /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     94..116
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     113..129
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     113..129
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     148..168
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        150..168
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        196..422
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             423..603
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    650..775
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1268..1501
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1686..1730
                     /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        1908..2156
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1908..1928
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2079..2098
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2312..2429
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2498..2709
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2735..3334
                     /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(2735..2754)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3231..3251
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3350..3938
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3403..3423)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3821..3832)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4010..4243
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4321..4442
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4358..4377)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4412..4431
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4482..4617
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4637..4656)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4638..4656)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4666..4683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4666..4682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4675..4697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4824..5279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4911..4930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5121..5142
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5305..5409
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5410..5478,5479..6270)
                     /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(5628..5647)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6441..7029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6930..6949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7183..7200
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
       61 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      121 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
      181 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga
      241 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag
      301 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac
      361 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa
      421 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
      481 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
      541 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
      601 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
      661 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
      721 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
      781 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
      841 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
      901 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
      961 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     1021 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     1081 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     1141 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     1201 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     1261 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     1321 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     1381 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     1441 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     1501 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     1561 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     1621 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     1681 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     1741 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     1801 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     1861 caccattatc gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc
     1921 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     1981 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2041 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2101 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcaa
     2161 gatgctatta taaggtggtt taagagctat gctggaaaca gcatagcaag tttaaataag
     2221 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattctaga
     2281 tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     2341 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     2401 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     2461 tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     2521 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     2581 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2641 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     2701 agaacacagg tgtcgtgacg cgggatccgc caccatgacc gagtacaagc ccacggtgcg
     2761 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     2821 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     2881 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     2941 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     3001 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     3061 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     3121 agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     3181 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     3241 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     3301 cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct
     3361 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3421 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3481 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     3541 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     3601 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     3661 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3721 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     3781 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3841 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3901 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact
     3961 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     4021 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     4081 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     4141 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     4201 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca
     4261 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     4321 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4381 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4441 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     4501 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4561 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4621 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     4681 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4741 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4801 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4861 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4921 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4981 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5041 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5101 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5161 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5221 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5281 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5341 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5401 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5461 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5521 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5581 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5641 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5701 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5761 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5821 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5881 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5941 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6001 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6061 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6121 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6181 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6241 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6301 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6361 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6421 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6481 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6541 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6601 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6661 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6721 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6781 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6841 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6901 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6961 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7021 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7081 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7141 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7201 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     7261 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gt
//
