LOCUS       sequence-337579-        5955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for CRISPR/Cas9 targeting of TERT RE2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5955)
  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 5955)
  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 RE2 Donor pLox hPGK-Puro pA pLox
FEATURES             Location/Qualifiers
     source          1..5955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             153..752
                     /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(153..172)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     649..669
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    761..882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(798..817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     852..871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(890..923)
                     /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(1569..1588)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1570..1588)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1595..1612)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1595..1611)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1604..1626)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1749..2051
                     /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     1930..1949
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2400..3194
                     /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             2400..3194
                     /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(2454..2473)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3064..3083
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3394..3403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3400..3774
                     /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      3912..4500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4401..4420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4654..4671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4788..4809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4824..4829,4830..4847,4848..4854)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4862..4878
                     /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     4867..4889
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4886..4902
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4886..4902
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4920..4938
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4920..4937
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    complement(5561..5594)
                     /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(5639..5662)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
       61 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
      121 tccatctaga tctcgagcag ctgaagctta ccatgaccga gtacaagccc acggtgcgcc
      181 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
      241 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
      301 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
      361 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
      421 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
      481 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
      541 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
      601 cggccgagcg cgccggggtg cccgccttcc tggaaacctc cgcgccccgc aacctcccct
      661 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
      721 cctggtgcat gacccgcaag cccggtgcct gagcggccgc aacttgttta ttgcagctta
      781 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
      841 gcattctagt tgtggtttgt ccaaactcat caatgtatct taactagtga taacttcgta
      901 tagcatacat tatacgaagt tatgaattct gcagaattcg cccttaaaaa actgcaggac
      961 caaaaccttt gtctttcatg tgagttatct gtgactgaaa gccttctttc tctctcccct
     1021 gagttaccat ttatgactaa aaccgttctc ttactgcctc tgggtcattt tttactaaaa
     1081 cctttatcac ctgagctatc acttgtgact gaaaaccttg tccatcacct gagctattta
     1141 gttttgattg aaacatcttt tctttgtcat acctacaaga atcataaatt atcctttatg
     1201 acttgcagct tcatctgtca atttagttat catttatgag tgaattcctt ttatctttat
     1261 cagctgagct atcatatatg gctggaaaac tttcttctct ctcgcctgag ttacctgtaa
     1321 tggaaggctt ctctgtatta ctggacttat ttacaactac agcgtctgtc tctctagcac
     1381 ttgagttatc atttatttat gactggccac cccgcatctc tctcacctgg tatcatttat
     1441 gtctaaaacc tttcattctc tatcaactga attattactg gaaaatgtct ttccccatca
     1501 tctagttgtc attcatgatt cagaccgttt tcagtcacct cagttccttg tggctggggc
     1561 ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg
     1621 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     1681 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctat
     1741 acgtacggca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg
     1801 atgtacagag tgatattatt gacacgccgg ggcgacggat ggtgatcccc ctggccagtg
     1861 cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg
     1921 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag
     1981 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct
     2041 ggggaatata aatgtcaggc atgagattat caaaaaggat cttcacctag atccttttca
     2101 cgtagaaagc cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta
     2161 tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat
     2221 ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg
     2281 cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa
     2341 ggatctgatg gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca
     2401 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     2461 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     2521 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     2581 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     2641 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     2701 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     2761 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     2821 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     2881 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     2941 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3001 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3061 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3121 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3181 acgagttctt ctgaattatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     3241 atctgtgcgg tatttcacac cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3301 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3361 tgataaatgc ttcaataata gcacgtgagg agggccacca tggccaagtt gaccagtgcc
     3421 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
     3481 gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
     3541 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
     3601 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
     3661 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
     3721 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg
     3781 ctaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3841 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3901 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3961 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4021 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4081 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4141 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4201 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4261 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4321 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4381 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4441 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4501 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4561 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4621 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4681 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     4741 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     4801 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     4861 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcta
     4921 tttaggtgac actatagaat actcaagcta tgcatcaagc ttggtacctg accacagaga
     4981 gagatacctg gtaaacaatg ctgacagtca cggaaggctg tgggcgactg ctgggcaccc
     5041 gttcctcagc gtggcctcag aagagctgcc atctagagcc acgcctgtgc cctgttccct
     5101 cctggcccct ccgcctccca ctgcagtcaa ggccccatct gcaccatttg ccatgacatg
     5161 cacagggtga tccatacctt tcccactgtg ccctgtggtc actggtcagg gatgaggtca
     5221 cgtcctcaga cctcagggac acagctgctg aggtcatggt ccttaaggaa ggccctgcca
     5281 gagtggcctg gggatgtcca ggcagttcca gaaggagtcc agagcggctg cccttgagaa
     5341 ggagatgggt gtgccctgag gaggggcagg agaagggggt gctggggggc tctaaggcag
     5401 ggccagcccc caggtcccca ggcatgggcc tgaggaccgg aaccaacttg cacccaggcc
     5461 ctcccagcca atgcccagcc cctggcactg ctgcctgtga gtgaggacag tgatgccgag
     5521 ctcggatcca ctagtaacgg ccgccagtgt gctggaattc ataacttcgt atagcataca
     5581 ttatacgaag ttatcattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     5641 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     5701 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     5761 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     5821 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     5881 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     5941 ctgggctcag gggcg
//
