LOCUS       sequence-128451-        6406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologues recombination donor plasmid for inserting a FLAG-SNAP-tag
            at the N-terminus of the TERT locus in human cells. Includes PURO 
            resistance flanked by LoxP sites for selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6406)
  AUTHORS   Xi L, Schmidt JC, Zaug AJ, Ascarrunz DR, Cech TR
  TITLE     A novel two-step genome editing strategy with CRISPR-Cas9 provides 
            new insights into telomerase action and TERT gene expression.
  JOURNAL   Genome Biol. 2015 Nov 10;16(1):231. doi: 10.1186/s13059-015-0791-1.
  PUBMED    26553065
REFERENCE   2  (bases 1 to 6406)
  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: "Genome 
            Biol."; date: "2015-11-10"; volume: "16(1):231. doi"; pages: " 
            10.1186/s13059-015-0791-1"
COMMENT     Sequence Label: FLAG-LoxP-PURO-LoxP-SNAP-TERT
FEATURES             Location/Qualifiers
     source          1..6406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1482..1547
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1548..1581
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1588..1636
                     /note="synthetic polyadenylation signal"
     CDS             complement(1637..2236)
                     /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(1720..1740)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2217..2236
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(2243..2559)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2257..2392
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    2566..2599
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2608..3153
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(4230..4246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4230..4246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4243..4265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4254..4270
                     /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(4278..4284,4285..4302,4303..4308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4323..4344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4461..4478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4632..5220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4712..4731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5391..6206)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5391..6206)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6114..6133
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6207..6311)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6379..6397
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggaac
      421 agtggccgtg tggcttctac tgctgggctg gaagtcgggc ctcctagctc tgcagtccga
      481 ggcttggagc caggtgcctg gaccccgagg ttgccctcca ccctgtgcgg gcgggatgtg
      541 accagatgtt ggcctcatct gccagacaga gtgccggggc ccagggtcaa ggccgttgtg
      601 gctggtgtga ggcgcccggt gcgcggccag caggagcgcc tggctccatt tcccaccctt
      661 tctcgacggg accgccccgg tgggtgatta acagatttgg ggtggtttgc tcatggtggg
      721 gacccctcgc cgcctgagaa cctgcaaaga gaaatgacgg gcctgtgtca aggagcccaa
      781 gtcgcgggga agtgttgcag ggaggcactc cgggaggtcc cgcgtgcccg tccagggagc
      841 aatgcgtcct cgggttcgtc cccagccgcg tctacgcgcc tccgtcctcc ccttcacgtc
      901 cggcattcgt ggtgcccgga gcccgacgcc ccgcgtccgg acctggaggc agccctgggt
      961 ctccggatca ggccagcggc caaagggtcg ccgcacgcac ctgttcccag ggcctccaca
     1021 tcatggcccc tccctcgggt taccccacag cctaggccga ttcgacctct ctccgctggg
     1081 gccctcgctg gcgtccctgc accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg
     1141 cccagacccc cgggtccgcc cggagcagct gcgctgtcgg ggccaggccg ggctcccagt
     1201 ggattcgcgg gcacagacgc ccaggaccgc gcttcccacg tggcggaggg actggggacc
     1261 cgggcacccg tcctgcccct tcaccttcca gctccgcctc ctccgcgcgg accccgcccc
     1321 gtcccgaccc ctcccgggtc cccggcccag ccccctccgg gccctcccag cccctcccct
     1381 tcctttccgc ggccccgccc tctcctcgcg gcgcgagttt caggcagcgc tgcgtcctgc
     1441 tgcgcacgtg ggaagccctg gccccggcca cccccgcgat ggactacaaa gaccatgacg
     1501 gtgattataa agatcatgat atcgattaca aggatgacga tgacaagata acttcgtata
     1561 atgtatgcta tacgaagtta taagcttcac acaaaaaacc aacacacaga tgtaatgaaa
     1621 ataaagatat tttatttcag gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt
     1681 cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag gggaggttgc
     1741 ggggcgcgga ggtctccagg aaggcgggca ccccggcgcg ctcggccgcc tccactccgg
     1801 ggagcacgac ggcgctgccc agacccttgc cctggtggtc gggcgagacg ccgacggtgg
     1861 ccaggaacca cgcgggctcc ttgggccggt gcggcgccag gaggccttcc atctgttgct
     1921 gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg
     1981 cccccgcttc gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga
     2041 cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc agctcggtga
     2101 cccgctcgat gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg
     2161 cggcggcgag ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg
     2221 gcttgtactc ggtcatgagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
     2281 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta
     2341 gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag
     2401 ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     2461 ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca
     2521 tacttctgcc tgctggggag cctggggact ttccacaccg gatccataac ttcgtataat
     2581 gtatgctata cgaagttatc cggtaccatg gacaaagatt gcgaaatgaa acgtaccacc
     2641 ctggatagcc cgctgggcaa actggaactg agcggctgcg aacagggcct gcatgaaatt
     2701 aaactgctgg gtaaaggcac cagcgcggcc gatgcggttg aagttccggc cccggccgcc
     2761 gtgctgggtg gtccggaacc gctgatgcag gcgaccgcgt ggctgaacgc gtattttcat
     2821 cagccggaag cgattgaaga atttccggtt ccggcgctgc atcatccggt gtttcagcag
     2881 gagagcttta cccgtcaggt gctgtggaaa ctgctgaaag tggttaaatt tggcgaagtg
     2941 attagctatc agcagctggc ggccctggcg ggtaatccgg cggccaccgc cgccgttaaa
     3001 accgcgctga gcggtaaccc ggtgccgatt ctgattccgt gccatcgtgt ggttagctct
     3061 agcggtgcgg ttggcggtta tgaaggtggt ctggcggtga aagagtggct gctggcccat
     3121 gaaggtcatc gtctgggtaa accgggtctg ggacctgcag gtataggcgc gccaggatcc
     3181 ggtaccatgc cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac
     3241 cgcgaggtgc tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg
     3301 gtgcagcgcg gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg
     3361 ccctgggacg cacggccgcc ccccgccgcc ccctccttcc gccaggtggg cctccccggg
     3421 gtcggcgtcc ggctggggtt gagggcggcc ggggggaacc agcgacatgc ggagagcagc
     3481 gcaggcgact cagggcgctt cccccgcagg tgtcctgcct gaaggagctg gtggcccgag
     3541 tgctgcagag gctgtgcgag cgcggcgcga agaacgtgct ggccttcggc ttcgcgctgc
     3601 tggacggggc ccgcgggggc ccccccgagg ccttcaccac cagcgtgcgc agctacctgc
     3661 ccaacacggt gaccgacgca ctgcggggga gcggggcgtg ggggctgctg ctgcgccgcg
     3721 tgggcgacga cgtgctggtt cacctgctgg cacgctgcgc gctctttgtg ctggtggctc
     3781 ccagctgcgc ctaccaggtg tgcgggccgc cgctgtacca gctcggcgct gccactcagg
     3841 cccggccccc gccacacgct agtggacccc gaaggcgtct gggatgcgaa cgggcctgga
     3901 accatagcgt cagggaggcc ggggtccccc tgggcctgcc agccccgggt gcgaggaggc
     3961 gcgggggcag tgccagccga agtctgccgt tgcccaagag gcccaggcgt ggcgctgccc
     4021 ctgagccgga gcggacgccc gttgggcagg ggtcctgggc ccacccgggc aggacgcgtg
     4081 gaccgagtga ccgtggtttc tgtgtggtgt cacctgccag acccgccgaa gaagccacct
     4141 ctttggaggg tgcgctctct ggcacgcgcc actcccaccc atccgatatc ggaacatccc
     4201 aatggcgcgc cgagcttggc tcgagcatgg tcatagctgt ttcctgtgtg aaattgttat
     4261 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4321 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4381 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4441 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4501 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4561 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4621 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4681 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4741 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4801 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4861 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4921 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4981 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5041 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5101 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5161 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5221 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5281 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5341 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttagaaaaac
     5401 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     5461 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     5521 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     5581 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     5641 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     5701 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     5761 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     5821 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     5881 acctggaatg ctgttttccc agggatcgca gtggtgagta accatgcatc atcaggagta
     5941 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     6001 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     6061 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     6121 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     6181 gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg aagcatttat
     6241 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6301 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6361 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
