LOCUS       sequence-310757-        6263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 6x His tagged TagRFP-T_A1aY1 in mammalian cells for 
            detection of tyrosinated microtubules..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6263)
  AUTHORS   Kesarwani S, Lama P, Chandra A, Reddy PP, Jijumon AS, Bodakuntla S, 
            Rao BM, Janke C, Das R, Sirajuddin M
  TITLE     Genetically encoded live-cell sensor for tyrosinated microtubules.
  JOURNAL   J Cell Biol. 2020 Oct 5;219(10). pii: 152071. doi: 
            10.1083/jcb.201912107.
  PUBMED    32886100
REFERENCE   2  (bases 1 to 6263)
  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: "J Cell 
            Biol."; date: "2020-10-5"; volume: "219(10). pii: 152071. doi"; 
            pages: " 10.1083/jcb.201912107"
COMMENT     Sequence Label: 6xHis-TagRFP-T_A1aY1
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      709..718
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             727..744
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             754..1482
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     intron          1770..1999
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1982..2001
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2072..2645
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2239..2256)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2466..2485
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2681..3484
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2744..2763)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3354..3373
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3516..3533)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3522..3746
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3793..3812
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3872..3888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3872..3888)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3885..3907)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3896..3912
                     /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(3920..3926,3927..3944,3945..3950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3965..3986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4103..4120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4274..4862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4354..4373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5033..5824,5825..5893))
                     /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     5656..5675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5894..5998)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6073..6092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcgat atctgcggcc tagctagcgc taccggtcgc caccatgggc
      721 agctcccatc atcatcatca tcacagctcc ggcgtgtcta agggcgaaga gctgattaag
      781 gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc
      841 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
      901 gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc
      961 agaaccttca tcaaccacac ccagggcatc cccgatttct ttaagcagtc cttccctgag
     1021 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     1081 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     1141 ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa caccgagatg
     1201 ctgtaccccg ctgacggcgg cctggaaggc agaaccgaca tggccctgaa gctcgtgggc
     1261 gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac
     1321 ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa ggaggccgac
     1381 aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc
     1441 aaactggggc acaaacttaa tggcatggac gagctgtaca agtccggact cagatctcga
     1501 gtggcaacag tcaagttcaa atacaagggg gaggaaaagc aggtggacat tagtaagatt
     1561 ctgagcgtcg gaagatacgg gaagctgatc cacttcctgt acgacctggg aggaggcaag
     1621 gcaggaatgg gcatggtgag cgagaaggat gcccccaagg agctgctcca gatgctggag
     1681 aagcagaaga agtaaggatc cgcggccgca tagataactg atccagtgtg ctggaattaa
     1741 ttcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctcaaaag cgggcatgac
     1801 ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca cctggcccgc
     1861 ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct ttttgttgtc
     1921 aagcttgagg tgtggcaggc ttgagatctg gccatacact tgagtgacaa tgacatccac
     1981 tttgcctttc tctccacagg tgtccactcc caggtccaac tgcaggtcga gcatgcatct
     2041 agggcggcca attccgcccc tctccccccc cccccyctcc ctcccccccc cctaacgtta
     2101 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     2161 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     2221 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     2281 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     2341 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     2401 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     2461 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     2521 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     2581 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     2641 gataagcttg ccacaacccg ggataattcc tgcagccaat atgggatcgg ccattgaaca
     2701 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     2761 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     2821 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     2881 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     2941 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3001 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3061 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3121 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3181 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgatgatct
     3241 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3301 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3361 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     3421 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     3481 ctgaggggat caattctcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     3541 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3601 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3661 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3721 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgag
     3781 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     3841 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3901 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3961 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4021 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4081 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4141 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4201 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4261 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4321 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4381 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4441 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4501 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4561 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4621 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4681 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4741 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4801 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4861 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4921 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4981 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5041 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5101 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5161 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5221 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5281 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5341 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5401 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5461 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5521 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5581 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5641 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5701 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5761 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5821 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5881 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5941 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6001 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6061 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6121 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     6181 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     6241 tgtacgggcc agatatacgc gtt
//
