LOCUS       sequence-388908-        5883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for CRISPR/Cas9 targeting of TERT Ex2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5883)
  AUTHORS   Laprade H, Querido E, Smith MJ, Guerit D, Crimmins H, Conomos D, 
            Pourret E, Chartrand P, Sfeir A
  TITLE     Single-Molecule Imaging of Telomerase RNA Reveals a 
            Recruitment-Retention Model for Telomere Elongation.
  JOURNAL   Mol Cell. 2020 Jul 2;79(1):115-126.e6. doi: 
            10.1016/j.molcel.2020.05.005. Epub 2020 Jun 3.
  PUBMED    32497497
REFERENCE   2  (bases 1 to 5883)
  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.molcel.2020.05.005"; journalName: "Mol Cell"; date: 
            "2020-07-2- 2"; volume: "79"; issue: "1"; pages: "115-126.e6"
COMMENT     Sequence Label: pTopo_Ex2Donor_pPGK-PURO
FEATURES             Location/Qualifiers
     source          1..5883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    101..122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(137..142,143..160,161..167)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    175..191
                     /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     180..202
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     199..215
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     199..215
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        233..251
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     233..250
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        882..1381
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(917..940)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1314..1333
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1421..2020
                     /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(1421..1440)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1917..1937
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2029..2150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2066..2085)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2120..2139
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2765..2784)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2766..2784)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2791..2808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2791..2807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2800..2822)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2945..3247
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3126..3145
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3596..4390
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3596..4390
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3650..3669)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4260..4279
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4590..4599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4596..4970
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      5108..5696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5597..5616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5850..5867
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
       61 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      121 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      181 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctatttaggt
      241 gacactatag aatactcaag ctatgcatca agcttggtac cgagctcgga tccactagta
      301 acggccgcca gtgtgctgga attcgccctt gtggcccagt gcctggtgtg cgtgccctgg
      361 gacgcacggc cgccccccgc cgccccctcc ttccgccagg tgggcctccc cggggtcggc
      421 gtccggctgg ggttgagggc ggccgggggg aaccagcgac atgcggagag cagcgcaggc
      481 gactcagggc gcttcccccg caggtgtcct gcctgaagga gctggtggcc cgagtgctgc
      541 agaggctgtg cgagcgcggc gcgaagaacg tgctggcctt cggcttcgcg ctgctggacg
      601 gggcccgcgg gggccccccc gaggccttca ccaccagcgt aagcagctac ctgcccaaca
      661 cggtgaccga cgcactgcgg gggagcgggg cgtgggggct gctgctgcgc cgcgtgggcg
      721 acgacgtgct ggttcacctg ctggcacgct gcgcgctctt tgtgctggtg gctcccagct
      781 gcgcctacca ggtgtgcggg ccgccgctgt accagctcgg cgctgccact caggcccggc
      841 ccccgccaca cgcaaaaggg tcccaaatga atgaatgaac cgggtagggg aggcgctttt
      901 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
      961 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1021 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     1081 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     1141 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     1201 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     1261 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     1321 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     1381 gacctgcatc catctagatc tcgagcagct gaagcttacc atgaccgagt acaagcccac
     1441 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     1501 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     1561 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     1621 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     1681 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     1741 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     1801 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     1861 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gaaacctccg cgccccgcaa
     1921 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     1981 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga gcggccgcaa cttgtttatt
     2041 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2101 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta gggtcccttt
     2161 ttagtggacc ccgaaggcgt ctgggatgcg aacgggcctg gaaccatagc gtcagggagg
     2221 ccggggtccc cctgggcctg ccagccccgg gtgcgaggag gcgcgggggc agtgccagcc
     2281 gaagtctgcc gttgcccaag aggcccaggc gtggcgctgc ccctgagccg gagcggacgc
     2341 ccgttgggca ggggtcctgg gcccacccgg gcaggacgcg tggaccgagt gaccgtggtt
     2401 tctgtgtggt gtcacctgcc agacccgccg aagaagccac ctctttggag ggtgcgctct
     2461 ctggcacgcg ccactcccac ccatccgtgg gccgccagca ccacgcgggc cccccatcca
     2521 catcgcggcc accacgtccc tgggacacgc cttgtccccc ggtgtacgcc gagaccaagc
     2581 acttcctcta ctcctcaggc gacaaggagc agctgcggcc ctccttccta ctcagctctc
     2641 tgaggcccag cctgactggc gctcggaggc tcgtggagac catctttctg ggttcaaggg
     2701 cgaattctgc agatatccat cacactggcg gccgctcgag catgcatcta gagggcccaa
     2761 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     2821 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     2881 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctatacgt
     2941 acggcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     3001 acagagtgat attattgaca cgccggggcg acggatggtg atccccctgg ccagtgcacg
     3061 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     3121 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     3181 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     3241 aatataaatg tcaggcatga gattatcaaa aaggatcttc acctagatcc ttttcacgta
     3301 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
     3361 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
     3421 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
     3481 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     3541 ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgcatgat
     3601 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3661 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3721 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     3781 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3841 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3901 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3961 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4021 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4081 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4141 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4201 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4261 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4321 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4381 gttcttctga attattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     4441 gtgcggtatt tcacaccgca tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4501 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4561 aaatgcttca ataatagcac gtgaggaggg ccaccatggc caagttgacc agtgccgttc
     4621 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     4681 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     4741 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     4801 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     4861 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     4921 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctaa
     4981 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5221 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5701 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5761 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5821 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5881 gcc
//
