LOCUS       Exported               11084 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian expression of 3xFLAG tagged TERT, N-terminal..
ACCESSION   .
VERSION     .
KEYWORDS    pCDH-3xFLAG-TERT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11084)
  AUTHORS   Zhong FL, Batista LF, Freund A, Pech MF, Venteicher AS, Artandi SE
  TITLE     TPP1 OB-fold domain controls telomere maintenance by recruiting 
            telomerase to chromosome ends.
  JOURNAL   Cell. 2012 Aug 3;150(3):481-94. doi: 10.1016/j.cell.2012.07.012.
  PUBMED    22863003
REFERENCE   2  (bases 1 to 11084)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2012.07"; journalName: "Cell"; date: "2012-08-3- 3";
            volume: "150"; issue: "3"; pages: "481-94"
FEATURES             Location/Qualifiers
     source          1..11084
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     201..225
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             305..370
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             380..3775
                     /codon_start=1
                     /gene="TERT"
                     /product="human telomerase reverse transcriptase, the 
                     catalytic subunit of telomerase"
                     /label=hTERT
                     /note="prevents telomere shortening and allows 
                     postsenescent cells to proliferate beyond crisis (Counter 
                     et al., 1998); also known as EST2"
                     /translation="PRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAA
                     FRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLD
                     GARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAP
                     SCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARR
                     RGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEA
                     TSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQL
                     RPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNH
                     AQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPW
                     QVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCA
                     WLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYR
                     KSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNM
                     DYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVL
                     RVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGH
                     VRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFM
                     CHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLL
                     VTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWC
                     GLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQ
                     VNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKA
                     KNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLP
                     GTTLTALEAAANPALPSDFKTILD"
     promoter        3830..4041
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             4054..4322
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             4350..5144
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4404..4423)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5014..5033
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    5151..5739
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5204..5224)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5622..5633)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5813..6046
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6118..6239
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6155..6174)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6209..6228
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6279..6414
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6341..6360
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(6447..6463)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6447..6463)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6460..6482)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6471..6487
                     /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(6495..6525)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6540..6561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6678..6695)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6849..7437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6929..6948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7608..8468)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8231..8250
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8469..8573)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8641..8659
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8697..8719)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     8819..8838
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9032..9054
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9046..9063
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9047..9063
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        9078..9304
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9305..9485
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    9532..9657
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10150..10383
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             10568..10612
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    10879..10995
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"