LOCUS       sequence-183885-        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 E4.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    94..115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(130..135,136..153,154..160)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    168..184
                     /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     173..195
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     192..208
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     192..208
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     227..247
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        229..247
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        275..501
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             502..682
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    729..854
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1347..1580
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1765..1809
                     /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        1987..2235
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1987..2007
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2158..2177
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2391..2508
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2577..2788
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2814..3413
                     /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(2814..2833)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3310..3330
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3429..4017
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3482..3502)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3900..3911)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4089..4322
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4400..4521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4437..4456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4491..4510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4561..4696
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4716..4735)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4717..4735)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4745..4762)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4745..4761)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4754..4776)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4903..5358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4990..5009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5200..5221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5384..5488
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5489..5557,5558..6349)
                     /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(5707..5726)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6520..7108
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7009..7028
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7262..7279
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
       61 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
      121 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
      181 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
      241 ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag
      301 tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt
      361 gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg
      421 ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag
      481 tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc
      541 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
      601 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
      661 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
      721 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
      781 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
      841 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
      901 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
      961 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     1021 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     1081 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     1141 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg
     1201 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat
     1261 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg
     1321 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca
     1381 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct
     1441 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg
     1501 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac
     1561 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact
     1621 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg
     1681 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt
     1741 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg
     1801 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata
     1861 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
     1921 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
     1981 cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     2041 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     2101 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     2161 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     2221 aaaggacgaa acaccgatga cctttacact tgatgagttt aagagctatg ctggaaacag
     2281 catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     2341 gctttttttg aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga
     2401 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2461 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     2521 agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc
     2581 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     2641 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2701 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2761 tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatgaccg
     2821 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2881 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     2941 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     3001 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3061 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3121 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3181 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3241 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3301 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3361 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt
     3421 aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3481 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3541 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3601 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3661 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3721 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3781 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3841 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3901 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3961 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc
     4021 gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     4081 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     4141 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     4201 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     4261 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     4321 cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     4381 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     4441 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4501 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     4561 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4621 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4681 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     4741 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4801 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4861 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4921 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4981 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5041 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5101 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5161 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5221 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5281 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5341 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5401 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5461 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5521 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5581 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5641 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5701 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5761 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5821 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5881 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5941 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6001 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6061 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6121 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6181 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6241 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6301 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6361 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6421 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6481 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6541 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6601 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6661 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6721 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6781 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6841 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6901 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6961 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7021 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7081 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7141 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7201 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7261 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cg
//
