LOCUS       sequence-128452-        6545 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 GFP 
            marker flanked by LoxP sites for selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6545)
  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 6545)
  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-GFP-LoxP-SNAP-TERT
FEATURES             Location/Qualifiers
     source          1..6545
                     /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)."
     primer_bind     complement(1589..1609)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1594..1923
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1774..1909
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(1930..1932,1933..1935,1936..2649)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1930..1932,1933..1935,1936..2646)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1975..1996)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2236..2255)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2583..2604
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2650..2698
                     /note="synthetic polyadenylation signal"
     protein_bind    2705..2738
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2747..3292
                     /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(4369..4385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4369..4385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4382..4404)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4393..4409
                     /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(4417..4423,4424..4441,4442..4447))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4462..4483
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4600..4617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4771..5359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4851..4870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5530..6345)
                     /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(5530..6345)
                     /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     6253..6272
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6346..6450)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6518..6536
                     /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 taagctttgg aatgtgtgtc agttagggtg tggaaagtcc
     1621 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     1681 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     1741 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     1801 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     1861 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     1921 aaaaagctca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1981 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2041 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2101 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2161 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2221 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2281 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2341 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2401 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2461 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2521 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2581 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2641 tacaagtaaa ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtggg
     2701 atccataact tcgtataatg tatgctatac gaagttatcc ggtaccatgg acaaagattg
     2761 cgaaatgaaa cgtaccaccc tggatagccc gctgggcaaa ctggaactga gcggctgcga
     2821 acagggcctg catgaaatta aactgctggg taaaggcacc agcgcggccg atgcggttga
     2881 agttccggcc ccggccgccg tgctgggtgg tccggaaccg ctgatgcagg cgaccgcgtg
     2941 gctgaacgcg tattttcatc agccggaagc gattgaagaa tttccggttc cggcgctgca
     3001 tcatccggtg tttcagcagg agagctttac ccgtcaggtg ctgtggaaac tgctgaaagt
     3061 ggttaaattt ggcgaagtga ttagctatca gcagctggcg gccctggcgg gtaatccggc
     3121 ggccaccgcc gccgttaaaa ccgcgctgag cggtaacccg gtgccgattc tgattccgtg
     3181 ccatcgtgtg gttagctcta gcggtgcggt tggcggttat gaaggtggtc tggcggtgaa
     3241 agagtggctg ctggcccatg aaggtcatcg tctgggtaaa ccgggtctgg gacctgcagg
     3301 tataggcgcg ccaggatccg gtaccatgcc gcgcgctccc cgctgccgag ccgtgcgctc
     3361 cctgctgcgc agccactacc gcgaggtgct gccgctggcc acgttcgtgc ggcgcctggg
     3421 gccccagggc tggcggctgg tgcagcgcgg ggacccggcg gctttccgcg cgctggtggc
     3481 ccagtgcctg gtgtgcgtgc cctgggacgc acggccgccc cccgccgccc cctccttccg
     3541 ccaggtgggc ctccccgggg tcggcgtccg gctggggttg agggcggccg gggggaacca
     3601 gcgacatgcg gagagcagcg caggcgactc agggcgcttc ccccgcaggt gtcctgcctg
     3661 aaggagctgg tggcccgagt gctgcagagg ctgtgcgagc gcggcgcgaa gaacgtgctg
     3721 gccttcggct tcgcgctgct ggacggggcc cgcgggggcc cccccgaggc cttcaccacc
     3781 agcgtgcgca gctacctgcc caacacggtg accgacgcac tgcgggggag cggggcgtgg
     3841 gggctgctgc tgcgccgcgt gggcgacgac gtgctggttc acctgctggc acgctgcgcg
     3901 ctctttgtgc tggtggctcc cagctgcgcc taccaggtgt gcgggccgcc gctgtaccag
     3961 ctcggcgctg ccactcaggc ccggcccccg ccacacgcta gtggaccccg aaggcgtctg
     4021 ggatgcgaac gggcctggaa ccatagcgtc agggaggccg gggtccccct gggcctgcca
     4081 gccccgggtg cgaggaggcg cgggggcagt gccagccgaa gtctgccgtt gcccaagagg
     4141 cccaggcgtg gcgctgcccc tgagccggag cggacgcccg ttgggcaggg gtcctgggcc
     4201 cacccgggca ggacgcgtgg accgagtgac cgtggtttct gtgtggtgtc acctgccaga
     4261 cccgccgaag aagccacctc tttggagggt gcgctctctg gcacgcgcca ctcccaccca
     4321 tccgatatcg gaacatccca atggcgcgcc gagcttggct cgagcatggt catagctgtt
     4381 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4441 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4501 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4561 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4621 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4681 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4741 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4801 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4861 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4921 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4981 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5041 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5101 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5161 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5221 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5281 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5341 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5401 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5461 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5521 gtctgacagt tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     5581 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     5641 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     5701 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     5761 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     5821 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     5881 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     5941 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     6001 atcaggatat tcttctaata cctggaatgc tgttttccca gggatcgcag tggtgagtaa
     6061 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     6121 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     6181 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     6241 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     6301 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca
     6361 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6421 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6481 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     6541 tcgtc
//
