LOCUS       sequence-170454-        7959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Telomere-targeting Nme sgRNA under U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7959)
  AUTHORS   Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, 
            Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR
  TITLE     Naturally Occurring Off-Switches for CRISPR-Cas9.
  JOURNAL   Cell. 2016 Dec 15;167(7):1829-1838.e9. doi: 
            10.1016/j.cell.2016.11.017. Epub 2016 Dec 8.
  PUBMED    27984730
REFERENCE   2  (bases 1 to 7959)
  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.cell.2016.11.017"; journalName: "Cell"; date: 
            "2016-12-15- 15"; volume: "167"; issue: "7"; pages: "1829-1838.e9"
COMMENT     Sequence Label: pEJS468-pLK.O1-NmeSgRNA/DTS13-Telomere
FEATURES             Location/Qualifiers
     source          1..7959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    52..173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(89..108)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     143..162
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      213..348
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(368..387)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(369..387)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(397..414)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(397..413)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(406..428)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      555..1010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(642..661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     852..873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1036..1140
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1141..1209,1210..2001)
                     /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(1359..1378)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2172..2760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2661..2680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2914..2931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3048..3069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  3094..3861
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(3862..3866,3867..3884,3885..3891)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3899..3915
                     /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     3904..3926
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3923..3939
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3923..3939
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3958..3978
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3960..3978
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4006..4232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4233..4413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4460..4585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5078..5311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5496..5540
                     /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        5838..6086
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5838..5858
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6009..6028
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6140..6232
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     misc_feature    6281..6398
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6447..6957
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6876..6894
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             6979..7578
                     /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(6979..6998)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7475..7495
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             7706..7939
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
       61 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
      121 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
      181 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
      241 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
      301 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
      361 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
      421 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
      481 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
      541 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
      601 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      661 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      721 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      781 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      841 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
      901 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
      961 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1021 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1081 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1141 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1201 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1261 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1321 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1381 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1441 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1501 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1561 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1621 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     1681 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     1741 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     1801 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     1861 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     1921 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     1981 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2041 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2101 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2161 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2221 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2281 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2341 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2401 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2461 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2521 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2581 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2641 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2701 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2761 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2821 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2881 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2941 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     3001 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     3061 ctcactcatt aggcacccca ggctttacac tttggtaatg actccaactt attgatagtg
     3121 ttttatgttc agataatgcc cgatgacttt gtcatgcagc tccaccgatt ttgagaacga
     3181 cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga ttcaggttat gccgctcaat
     3241 tcgctgcgta tatcgcttgc tgattacgtg cagctttccc ttcaggcggg attcatacag
     3301 cggccagcca tccgtcatcc atatcaccac gtcaaagggt gacagcaggc tcataagacg
     3361 ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca acaaccgtct tccggagact
     3421 gtcatacgcg taaaacagcc agcgctggcg cgatttagcc ccgacatagc cccactgttc
     3481 gtccatttcc gcgcagacga tgacgtcact gcccggctgt atgcgcgagg ttaccgactg
     3541 cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc caacgcccat aatgcgggct
     3601 gttgcccggc atccaacgcc attcatggcc atatcaatga ttttctggtg cgtaccgggt
     3661 tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac ggcagtgaga gcagagatag
     3721 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag
     3781 ggacagctga tagaaacaga agccactgga gcacctcaaa aacaccatca tacactaaat
     3841 cagtaagttg gcagcatcac cttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     3901 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
     3961 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
     4021 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
     4081 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
     4141 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
     4201 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
     4261 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     4321 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     4381 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
     4441 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
     4501 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
     4561 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
     4621 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
     4681 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     4741 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     4801 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     4861 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     4921 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     4981 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     5041 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     5101 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     5161 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     5221 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     5281 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     5341 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     5401 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     5461 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     5521 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     5581 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     5641 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     5701 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
     5761 acagatccat tcgattagtg aacggatctc gacggtatcg atcacgagac tagcctcgag
     5821 cggccgcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     5881 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     5941 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     6001 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     6061 atatcttgtg gaaaggacga aacaccgtta gggttagggt tagggttagg ggttgtagct
     6121 ccctttctca tttcggaaac gaaatgagaa ccgttgctac aataaggccg tctgaaaaga
     6181 tgtgccgcaa cgctctgccc cttaaagctt ctgctttaag gggcatcgtt tatttttttg
     6241 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     6301 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     6361 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     6421 gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct
     6481 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
     6541 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
     6601 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
     6661 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
     6721 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
     6781 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
     6841 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
     6901 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg
     6961 gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     7021 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     7081 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     7141 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     7201 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     7261 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     7321 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     7381 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     7441 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     7501 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     7561 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     7621 cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     7681 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     7741 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     7801 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     7861 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     7921 gtgtggaaaa tctctagcag tagtagttca tgtcatctt
//
