LOCUS       sequence-335427-        4859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty donor template for CRISPR/Cas9 targeting of TERT RE's.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4859)
  AUTHORS   Penev A, Bazley A, Shen M, Boeke JD, Savage SA, Sfeir A
  TITLE     Alternative splicing is a developmental switch for hTERT expression.
  JOURNAL   Mol Cell. 2021 Apr 6. pii: S1097-2765(21)00228-8. doi: 
            10.1016/j.molcel.2021.03.033.
  PUBMED    33852895
REFERENCE   2  (bases 1 to 4859)
  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"; journalName: "Mol Cell. 
            2021 Apr 6. pii: S1097-2765(21)00228-8. doi: 
            10.1016/j.molcel.2021.03.033."
COMMENT     Sequence Label: pTopo pLox hPGK-Puro pA pLox
FEATURES             Location/Qualifiers
     source          1..4859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     315..338
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    383..416
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(490..508)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(491..508)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(526..542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(526..542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(539..561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    550..566
                     /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)."
     promoter        complement(join(574..580,581..598,599..604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    619..640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(757..774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(928..1516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1008..1027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1654..2028)
                     /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"
     regulatory      2025..2034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2234..3028)
                     /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             complement(2234..3028)
                     /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(2345..2364)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2955..2974
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3377..3679)
                     /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     complement(3479..3498)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3802..3824
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3816..3833
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3817..3833
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3840..3859
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3840..3858
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3923..3956
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3964..4085
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3975..3994)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4029..4048
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4094..4693)
                     /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(4177..4197)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4674..4693
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(join(4733..4859,1..373))
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4781..4800)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 cccccggacc caccccttcc cagcctctga gcccagaaag cgaaggagca aagctgctat
       61 tggccgctgc cccaaaggcc tacccgcttc cattgctcag cggtgctgtc catctgcacg
      121 agactagtga gacgtgctac ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg
      181 ggggggaact tcctgactag gggaggagta gaaggtggcg cgaaggggcc accaaagaac
      241 ggagccggtt ggcgcctacc ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg
      301 tagcgccaag tgcccagcgg ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc
      361 gcctccccta cccggtagaa tgataacttc gtataatgta tgctatacga agttatgaat
      421 tccagcacac tggcggccgt tactagtgga tccgagctcg gtaccaagct tgatgcatag
      481 cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc
      541 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
      601 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
      661 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
      721 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
      781 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
      841 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
      901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     1321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     1381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1561 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1621 ccttttaaat taaaaatgaa gttttagcac gtgtcagtcc tgctcctcgg ccacgaagtg
     1681 cacgcagttg ccggccgggt cgcgcagggc gaactcccgc ccccacggct gctcgccgat
     1741 ctcggtcatg gccggcccgg aggcgtcccg gaagttcgtg gacacgacct ccgaccactc
     1801 ggcgtacagc tcgtccaggc cgcgcaccca cacccaggcc agggtgttgt ccggcaccac
     1861 ctggtcctgg accgcgctga tgaacagggt cacgtcgtcc cggaccacac cggcgaagtc
     1921 gtcctccacg aagtcccggg agaacccgag ccggtcggtc cagaactcga ccgctccggc
     1981 gacgtcgcgc gcggtgagca ccggaacggc actggtcaac ttggccatgg tggccctcct
     2041 cacgtgctat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2101 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     2161 tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
     2221 aagcgttaat aattcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     2281 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     2341 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     2401 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     2461 cgccatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca
     2521 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     2581 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     2641 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     2701 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     2761 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     2821 gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct
     2881 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     2941 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     3001 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     3061 agcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc
     3121 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc
     3181 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct
     3241 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc
     3301 accgtttctg cggactggct ttctacgtga aaaggatcta ggtgaagatc ctttttgata
     3361 atctcatgcc tgacatttat attccccaga acatcaggtt aatggcgttt ttgatgtcat
     3421 tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc acactggcca
     3481 tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg taaagttcac
     3541 gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc cgtcgccccg
     3601 gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc tctcttttat
     3661 aggtgtaaac cttaaactgc cgtacgtata ggctgcgcaa ctgttgggaa gggcgatcgg
     3721 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     3781 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     3841 aatacgactc actatagggc gaattgggcc ctctagatgc atgctcgagc ggccgccagt
     3901 gtgatggata tctgcagaat tcataacttc gtataatgta tgctatacga agttatcact
     3961 agttaagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     4021 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4081 aagttgcggc cgctcaggca ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg
     4141 gcacctcgac gtcggcggtg acggtgaagc cgagccgctc gtagaagggg aggttgcggg
     4201 gcgcggaggt ttccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga
     4261 gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg acggtggcca
     4321 ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg
     4381 cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc
     4441 ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc
     4501 acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc
     4561 gctcgatgtg gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg
     4621 cggcgagggt gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct
     4681 tgtactcggt catggtaagc ttcagctgct cgagatctag atggatgcag gtcgaaaggc
     4741 ccggagatga ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg
     4801 ccgggcctcc ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccg
//
