LOCUS       sequence-183160-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TBCB.
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: TBCB E12.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    85..106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(121..126,127..144,145..151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    159..175
                     /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     164..186
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     183..199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     183..199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     218..238
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        220..238
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        266..492
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             493..673
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    720..845
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1338..1571
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1756..1800
                     /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        1978..2226
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1978..1998
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2149..2168
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2382..2499
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2568..2779
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2805..3404
                     /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(2805..2824)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3301..3321
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3420..4008
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3473..3493)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3891..3902)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4080..4313
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4391..4512
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4428..4447)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4482..4501
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4552..4687
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4707..4726)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4708..4726)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4736..4753)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4736..4752)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4745..4767)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4894..5349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4981..5000)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5191..5212
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5375..5479
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5480..5548,5549..6340)
                     /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(5698..5717)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6511..7099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7000..7019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7253..7270
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
       61 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      121 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      181 cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga
      241 acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac
      301 atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga
      361 ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca
      421 tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct
      481 cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
      541 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
      601 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
      661 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
      721 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
      781 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
      841 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
      901 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
      961 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     1021 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     1081 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     1141 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
     1201 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     1261 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     1321 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     1381 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     1441 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     1501 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     1561 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     1621 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     1681 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     1741 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     1801 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     1861 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     1921 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccagagag
     1981 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     2041 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     2101 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     2161 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     2221 aacaccgcgc ccgccttggt gagtatggtt taagagctat gctggaaaca gcatagcaag
     2281 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     2341 gaattctaga tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     2401 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     2461 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     2521 agagatccac tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac
     2581 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     2641 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2701 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2761 gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc caccatgacc gagtacaagc
     2821 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     2881 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     2941 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3001 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3061 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3121 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3181 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3241 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3301 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3361 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagtcgaca
     3421 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3481 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3541 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3601 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3661 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3721 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3781 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3841 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3901 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3961 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag
     4021 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     4081 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     4141 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     4201 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     4261 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtacgta
     4321 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     4381 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4441 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4501 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     4561 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4621 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4681 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4741 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4801 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4861 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4921 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4981 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5041 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5101 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5161 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5221 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5281 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     5341 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5401 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5461 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5521 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5581 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5641 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5701 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5761 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5821 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5881 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5941 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6001 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6061 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6121 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6181 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6241 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6301 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6361 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6421 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6481 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6541 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6601 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6661 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6721 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6781 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6841 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6901 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6961 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7021 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7081 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7141 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     7201 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     7261 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cc
//
