LOCUS       sequence-369205-        6693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP tagged human 2N4R tau (with T217E mutation) in 
            mammalian cells with CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6693)
  AUTHORS   Rajbanshi B, Guruacharya A, Mandell JW, Bloom GS
  TITLE     Localization, induction, and cellular effects of tau phosphorylated 
            at threonine 217.
  JOURNAL   Alzheimers Dement. 2023 Jan 12. doi: 10.1002/alz.12892.
  PUBMED    36633254
REFERENCE   2  (bases 1 to 6693)
  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: "Alzheimers 
            Dement. 2023 Jan 12. doi: 10.1002/alz.12892."
COMMENT     Sequence Label: EGFP-Tau(T217E)
FEATURES             Location/Qualifiers
     source          1..6693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        1020..1399
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1400..1603
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1554..1574
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1600..1624
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1834..1852
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1834..1851
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1865..1884
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1928..1937
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1934..1936,1937..1939,1940..2650)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSNQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1979..2000)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2240..2259)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2587..2608
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3999..4008
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4012..4146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4049..4068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4103..4122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4215..4572
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4215..4406
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4238..4258)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4423..4558
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4592..4609)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4592..4608)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4601..4623)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4821..5276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4908..4927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5118..5139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5293..5312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5412..5434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5472..5490)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5558..5662
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5663..5731,5732..6523)
                     /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     complement(5881..5900)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga catgattacg aattaattcg agctcgcccg
     1021 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     1081 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1141 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1201 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1261 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1321 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1381 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1441 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1501 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1561 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     1621 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
     1681 cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt
     1741 aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa
     1801 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc
     1861 cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc
     1921 gatggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1981 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2041 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2101 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     2161 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2221 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2281 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2341 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2401 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2461 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2521 gcccgacaac cactacctga gcaaccagtc cgccctgagc aaagacccca acgagaagcg
     2581 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2641 gctgtacaag ggatccgctg agccccgcca ggagttcgaa gtgatggaag atcacgctgg
     2701 gacgtacggg ttgggggaca ggaaagatca ggggggctac accatgcacc aagaccaaga
     2761 gggtgacacg gacgctggcc tgaaagaatc tcccctgcag acccccactg aggacggatc
     2821 tgaggaaccg ggctctgaaa cctctgatgc taagagcact ccaacagcgg aagatgtgac
     2881 agcaccctta gtggatgagg gagctcccgg caagcaggct gccgcgcagc cccacacgga
     2941 gatcccagaa ggaaccacag ctgaagaagc aggcattgga gacaccccca gcctggaaga
     3001 cgaagctgct ggtcacgtga cccaagctcg catggtcagt aaaagcaaag acgggactgg
     3061 aagcgatgac aaaaaagcca agggggctga tggtaaaacg aagatcgcca caccgcgggg
     3121 agcagcccct ccaggccaga agggccaggc caacgccacc aggattccag caaaaacccc
     3181 gcccgctcca aagacaccac ccagctctgg tgaacctcca aaatcagggg atcgcagcgg
     3241 ctacagcagc cccggctccc caggcactcc cggcagccgc tcccgcaccc cgtcccttcc
     3301 agaaccaccc acccgggagc ccaagaaggt ggcagtggtc cgtactccac ccaagtcgcc
     3361 gtcttccgcc aagagccgcc tgcagacagc ccccgtgccc atgccagacc tgaagaatgt
     3421 caagtccaag atcggctcca ctgagaacct gaagcaccag ccgggaggcg ggaaggtgca
     3481 gataattaat aagaagctgg atcttagcaa cgtccagtcc aagtgtggct caaaggataa
     3541 tatcaaacac gtcccgggag gcggcagtgt gcaaatagtc tacaaaccag ttgacctgag
     3601 caaggtgacc tccaagtgtg gctcattagg caacatccat cataaaccag gaggtggcca
     3661 ggtggaagta aaatctgaga agcttgactt caaggacaga gtccagtcga agattgggtc
     3721 cctggacaat atcacccacg tccctggcgg aggaaataaa aagattgaaa cccacaagct
     3781 gaccttccgc gagaacgcca aagccaagac agaccacggg gcggagatcg tgtacaagtc
     3841 gccagtggtg tctggggaca cgtctccacg gcatctcagc aatgtctcct ccaccggcag
     3901 catcgacatg gtagactcgc cccagctcgc cacgctagct gacgaggtgt ctgcctccct
     3961 ggccaagcag ggtttgtgag tcgacctgca gaagcttggc cgccatggcc caacttgttt
     4021 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4081 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4141 tggatcggga attaattcgg cgcagcacca tggcctgaaa taacctctga aagaggaact
     4201 tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     4261 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4321 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4381 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4441 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4501 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4561 ggcttttgca aaaagctgtt aacagcttgg cactggccgt cgttttacaa cgtcgtgact
     4621 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     4681 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4741 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4801 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4861 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4921 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4981 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     5041 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5101 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5161 gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5221 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     5281 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5341 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5401 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5461 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     5521 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5581 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5641 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5701 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5761 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5821 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5881 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5941 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6001 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6061 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6121 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6181 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6241 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6301 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6361 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6421 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6481 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6541 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6601 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6661 gtcagacccc gtagaaaaga tcaaaggatc ttc
//
