LOCUS       sequence-337578-        5918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for CRISPR/Cas9 targeting of TERT RE3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  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 RE3 Donor pLox hPGK-Puro pA pLox
FEATURES             Location/Qualifiers
     source          1..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     144..163
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             251..850
                     /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(251..270)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     747..767
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    859..980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(896..915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     950..969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(988..1021)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1640..1659)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1641..1659)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1666..1683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1666..1682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1675..1697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1820..2122
                     /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     2001..2020
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2471..3265
                     /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             2471..3265
                     /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(2525..2544)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3135..3154
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3465..3474
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3471..3845
                     /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      3983..4571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4472..4491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4725..4742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4859..4880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4895..4900,4901..4918,4919..4925)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4933..4949
                     /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     4938..4960
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4957..4973
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4957..4973
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4991..5009
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4991..5008
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    complement(5622..5655)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5700..5723)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa
       61 tagcagcttt gctccttcgc tttctgggct caggggcggg ctcaggggcg gggcgggcgc
      121 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct
      181 gttctcctct tcctcatctc cgggcctttc gacctgcatc catctagatc tcgagcagct
      241 gaagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
      301 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
      361 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
      421 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
      481 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
      541 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
      601 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
      661 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
      721 cgccttcctg gaaacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
      781 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
      841 cggtgcctga gcggccgcaa cttgtttatt gcagcttata atggttacaa ataaagcaat
      901 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
      961 aaactcatca atgtatctta actagtgata acttcgtata gcatacatta tacgaagtta
     1021 tgaattctgc agatatccat cacactggcg gccgctcgag cacgcctggc tccaaggaag
     1081 ctggggagcc agggggtcta ggaacagtga gagatgctca tgttgacaaa gaatgtgccc
     1141 gccagccaca gagcagctcc gcccggtcct acactcaccc tgcatgcagc tccatcgctt
     1201 ctctcagtgc tggcggctcc agagcttctg gagctgcagg atgcttggaa agaacataga
     1261 ctcaaggaaa gtggcgcctg gagcctgcag ccactggtga gatgggtgga agcagccctc
     1321 agaggccgat ccctccctca caccatcagg atagtctgtt agcctcgcca ggccccagct
     1381 ttcttgtcca tggagtgggg ttactaacag ttcccacctc agaggctggg attcatagag
     1441 tctgggtttt aggggaggct gggatcagag aagctgggat cgggaggctg ggagtggggg
     1501 tcctgggatg gaggaggctg ggattaggag gctgggattg gggaggctgg gattgggagg
     1561 ctgggataga ggaggctggg attgagggca cctgggttcc ctctggtctc tgccttctcc
     1621 ctggctgggg cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     1681 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1741 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1801 gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     1861 tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc
     1921 cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     1981 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     2041 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     2101 cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta
     2161 gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag
     2221 ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag
     2281 tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt
     2341 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
     2401 cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg
     2461 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2521 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2581 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2641 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2701 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     2761 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     2821 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     2881 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     2941 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     3001 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3061 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3121 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3181 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3241 tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt
     3301 tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg
     3361 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3421 gacaataacc ctgataaatg cttcaataat agcacgtgag gagggccacc atggccaagt
     3481 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     3541 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     3601 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     3661 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     3721 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     3781 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     3841 actgacacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3901 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3961 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4021 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4081 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4141 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4201 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4261 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4321 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4381 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4441 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4501 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4561 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4621 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4681 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4741 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     4801 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     4861 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     4921 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     4981 acgccaagct atttaggtga cactatagaa tactcaagct atgcatcaag cttggtaccg
     5041 tgggcactgg ggcactgctg gtgccatctc ccacctcagg cccctccaag gggtgtccct
     5101 gggctgcttc ccctccctct gctggggggc tgccctctcc ctgaagcacc cccagtccct
     5161 ccccagcaga ctgatgccga gactgaggca gaggctgaaa gtcatttcca aaaggtcctc
     5221 ggtgacactg gaactcgttc tgcttgttaa aaatttcaaa ccggaacgag actttggagc
     5281 ttattttgtc caagcctttt atttttcaaa ggaggcaact gcactgagag cgttagactc
     5341 aaggtgcctg gcactgactc aggctccagg gcaccccctc cccgggatgc cccacacgaa
     5401 gcacccgtcc ctagaaggtg gccccaggtt cgtggtccca tcttcaaccc cagggtttac
     5461 tgtctctata ttctctgccg tgaagtctaa gggcagctca aatttcataa acggagaaac
     5521 agtcctattt gtcgattctc aactgccttt atctccagag atcccgtgag agcagggaga
     5581 gctcggatcc actagtaacg gccgccagtg tgctggaatt cataacttcg tatagcatac
     5641 attatacgaa gttatcattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     5701 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     5761 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     5821 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     5881 ggacgtgaca aatggaagta gcacgtctca ctagtctc
//
