LOCUS       sequence-174395-        6775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR Donor for N-terminal FLAG-HaloTag-TERT fusion in human cells. 
            Includes Puro selection marker flanked by LoxP sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6775)
  AUTHORS   Schmidt JC, Zaug AJ, Cech TR
  TITLE     Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to 
            Telomeres.
  JOURNAL   Cell. 2016 Aug 25;166(5):1188-1197.e9. doi: 
            10.1016/j.cell.2016.07.033. Epub 2016 Aug 11.
  PUBMED    27523609
REFERENCE   2  (bases 1 to 6775)
  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"; doi: 
            "10.1016/j.cell.2016.07.033"; journalName: "Cell"; date: 
            "2016-08-25- 25"; volume: "166"; issue: "5"; pages: "1188-1197.e9"
COMMENT     Sequence Label: FLAG-LoxP-PURO-LoxP-HaloTERT WT HR Donor
FEATURES             Location/Qualifiers
     source          1..6775
                     /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..3498
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             3511..3531
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(4599..4615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4599..4615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4612..4634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4623..4639
                     /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(4647..4653,4654..4671,4672..4677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4692..4713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4830..4847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5001..5589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5081..5100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5760..6575)
                     /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(5760..6575)
                     /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     6483..6502
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6576..6680)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6748..6766
                     /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 gcagaaatcg gtactggctt tccattcgac
     2641 ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat
     2701 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
     2761 atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa
     2821 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
     2881 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
     2941 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
     3001 atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc
     3061 ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt
     3121 acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag
     3181 ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc
     3241 gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag
     3301 tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa
     3361 gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg
     3421 aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg
     3481 acgctcgaga tttccggcga gccaaccact gaggatctgt actttcagag cgataacgcg
     3541 atcgcttccg gtaccatgcc gcgcgctccc cgctgccgag ccgtgcgctc cctgctgcgc
     3601 agccactacc gcgaggtgct gccgctggcc acgttcgtgc ggcgcctggg gccccagggc
     3661 tggcggctgg tgcagcgcgg ggacccggcg gctttccgcg cgctggtggc ccagtgcctg
     3721 gtgtgcgtgc cctgggacgc acggccgccc cccgccgccc cctccttccg ccaggtgggc
     3781 ctccccgggg tcggcgtccg gctggggttg agggcggccg gggggaacca gcgacatgcg
     3841 gagagcagcg caggcgactc agggcgcttc ccccgcaggt gtcctgcctg aaggagctgg
     3901 tggcccgagt gctgcagagg ctgtgcgagc gcggcgcgaa gaacgtgctg gccttcggct
     3961 tcgcgctgct ggacggggcc cgcgggggcc cccccgaggc cttcaccacc agcgtgcgca
     4021 gctacctgcc caacacggtg accgacgcac tgcgggggag cggggcgtgg gggctgctgc
     4081 tgcgccgcgt gggcgacgac gtgctggttc acctgctggc acgctgcgcg ctctttgtgc
     4141 tggtggctcc cagctgcgcc taccaggtgt gcgggccgcc gctgtaccag ctcggcgctg
     4201 ccactcaggc ccggcccccg ccacacgcta gtggaccccg aaggcgtctg ggatgcgaac
     4261 gggcctggaa ccatagcgtc agggaggccg gggtccccct gggcctgcca gccccgggtg
     4321 cgaggaggcg cgggggcagt gccagccgaa gtctgccgtt gcccaagagg cccaggcgtg
     4381 gcgctgcccc tgagccggag cggacgcccg ttgggcaggg gtcctgggcc cacccgggca
     4441 ggacgcgtgg accgagtgac cgtggtttct gtgtggtgtc acctgccaga cccgccgaag
     4501 aagccacctc tttggagggt gcgctctctg gcacgcgcca ctcccaccca tccgatatcg
     4561 gaacatccca atggcgcgcc gagcttggct cgagcatggt catagctgtt tcctgtgtga
     4621 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4681 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4861 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4921 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4981 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5041 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5101 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5161 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5221 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5281 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5341 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5401 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5461 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5521 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5581 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5641 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5701 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5761 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     5821 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     5881 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     5941 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     6001 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     6061 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     6121 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     6181 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     6241 tcttctaata cctggaatgc tgttttccca gggatcgcag tggtgagtaa ccatgcatca
     6301 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     6361 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     6421 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     6481 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     6541 ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga
     6601 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6661 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6721 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
