LOCUS       sequence-183884-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human POT1.
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: POT1 E3.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..89
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    206..227
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(242..247,248..265,266..272)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    280..296
                     /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     285..307
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     304..320
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     304..320
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     339..359
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        341..359
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        387..613
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             614..794
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    841..966
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1459..1692
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1877..1921
                     /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        2099..2347
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2099..2119
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2270..2289
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2503..2620
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2689..2900
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2926..3525
                     /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(2926..2945)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3422..3442
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3541..4129
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3594..3614)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4012..4023)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4201..4434
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4512..4633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4549..4568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4603..4622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4673..4808
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4828..4847)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4829..4847)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4857..4874)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4857..4873)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4866..4888)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5015..5470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5102..5121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5312..5333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5496..5600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5601..5669,5670..6461)
                     /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(5819..5838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6632..7220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7121..7140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
       61 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
      121 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
      181 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      241 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
      301 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
      361 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
      421 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
      481 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
      541 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
      601 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
      661 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
      721 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
      781 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
      841 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
      901 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
      961 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     1021 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     1081 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1141 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1201 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1261 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     1321 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1381 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1441 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1501 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1561 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1621 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1681 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     1741 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     1801 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     1861 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     1921 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     1981 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     2041 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga
     2101 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     2161 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     2221 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     2281 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     2341 aaacaccgag tactagaagc ctatctcagt ttaagagcta tgctggaaac agcatagcaa
     2401 gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     2461 tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2521 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2581 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2641 cagagatcca ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca
     2701 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     2761 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2821 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2881 ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag
     2941 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     3001 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     3061 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3121 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     3181 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     3241 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     3301 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     3361 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     3421 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     3481 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac
     3541 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3601 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3661 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3721 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3781 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3841 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3901 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3961 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     4021 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     4081 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     4141 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     4201 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     4261 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     4321 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     4381 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     4441 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     4501 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4561 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4621 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     4681 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4741 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4801 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     4861 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     4921 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     4981 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     5041 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5101 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5161 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5221 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5281 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5341 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5401 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5461 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5521 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5581 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5641 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5701 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5761 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5821 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5881 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5941 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6001 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6061 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6121 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6181 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6241 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6301 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6361 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6421 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6481 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6541 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6661 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6721 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6781 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6841 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6961 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7021 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7081 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7141 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7261 gctggccttt tgctcacatg ttctttcctg cgttatcccc tg
//
