LOCUS       sequence-434242-        6585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally EGFP-tagged IFT54 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6585)
  AUTHORS   Hiyamizu S, Qiu H, Vuolo L, Stevenson NL, Shak C, Heesom KJ, Hamada 
            Y, Tsurumi Y, Chiba S, Katoh Y, Stephens DJ, Nakayama K
  TITLE     Multiple interactions of the dynein-2 complex with the IFT-B complex
            are required for effective intraflagellar transport.
  JOURNAL   J Cell Sci. 2023 Mar 1;136(5):jcs260462. doi: 10.1242/jcs.260462. 
            Epub 2023 Feb 7.
  PUBMED    36632779
REFERENCE   2  (bases 1 to 6585)
  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 
            Sci."; date: "2023-03-1"; volume: "136(5):jcs260462. doi"; pages: " 
            10.1242/jcs.260462. Epub 2023 Feb 7"
COMMENT     Sequence Label: pEGFP-C1-IFT54
FEATURES             Location/Qualifiers
     source          1..6585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3373..3494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3410..3429)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3464..3483
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3501..3956)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3638..3659)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3850..3869
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3983..4087
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4089..4446
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4089..4280
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4112..4132)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4297..4432
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4481..5275
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4481..5275
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4535..4554)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5145..5164
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5463..5482)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5507..5554
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5883..6471
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6372..6391
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atccatgaac gcggcggtgg tgaggcggac gcaggaggcg
     1381 ctggggaaag tgattcggag gccgccgctg acagagaagc tgctgagcaa gcccccgttc
     1441 cgctacctgc acgacatcat cacggaggtg attagaatga ctggtttcat gaagggcctc
     1501 tacacagacg ccgagatgaa gtctgataat gtgaaggata aagatgcaaa aattagcttc
     1561 ctacaaaagg ccatagacgt ggttgtaatg gtgtcgggag agccactgtt ggccaaacca
     1621 gcccgaatcg tggcggggca tgagcctgaa agaacaaacg agctgctcca gataattgga
     1681 aaatgctgtc tcaacaagct ctctagtgac gatgcggtgc ggagggtttt agctggagag
     1741 aagggagaag tgaaaggccg ggcctcactg acctcaagat ctcaggaatt ggataataag
     1801 aatgtgcgag aagaagagtc cagagttcac aaaaatacag aggatagagg agacgctgaa
     1861 ataaaagaga gaagtacaag cagagatcga aaacagaagg aagaattgaa agaagaccgc
     1921 aagccaagag aaaaggacaa ggacaaggag aaggccaagg agaatggcgg aaacagacac
     1981 agagaagggg agagagagag agccaaagcc cgggccaggc cagacagcga gcgacagaaa
     2041 gacagaggca acagggagcg ggacagagac tccgagcgca agaaggagac agagagaaag
     2101 agtgaggggg ggaaagagaa ggagagactg agagacaggg accgagagcg cgaccgggac
     2161 aaagggaagg acagggacag acggagagtg aaaaacgggg agcactcctg ggacctggac
     2221 agggagaaga acagagagca tgacaaaccc gagaaaaagt cagcaagctc aggggagatg
     2281 tctaaaaagt tatcagatgg aacttttaaa gactccaagg ctgaaacaga gactgagatt
     2341 tccactagag cttccaagtc attgacaaca aaaacatcaa aacggcgatc caaaaattca
     2401 gtggaaggtg actccaccag tgatgcagaa ggagatgctg gacctgctgg ccaagataag
     2461 tctgaggtgc cagagactcc agaaattcct aatgagcttt catccaacat cagaagaatt
     2521 cctcggcctg ggagtgcaag accagcccct ccccgggtca aacggcaaga cagcatggag
     2581 gcgctacaaa tggataggtc agggagtggt aaaaccgttt caaatgtgat tacagagtca
     2641 cacaattctg acaatgaaga ggatgatcaa tttgtggtgg aagctgcccc tcagctctct
     2701 gaaatgtcag aaattgaaat ggtaacagca gtggaactag aagaagagga gaagcatggt
     2761 ggacttgtga aaaaaatttt ggagacgaag aaagattatg agaaattgca gcagtcaccc
     2821 aaacctgggg agaaggagcg atctctcttt gagtcggcat ggaagaagga gaaggacatc
     2881 gtttccaagg agatagagaa gctccgcacg tccatccaga ccctgtgcaa gagcgcactt
     2941 cccctgggga agatcatgga ctacatccag gaagacgtgg atgccatgca gaatgagctg
     3001 cagatgtggc acagcgagaa caggcagcac gccgaggccc tgcagcagga gcagaggatc
     3061 acagactgtg ccgtggagcc cttaaaggct gagctcgcgg agctggagca gctgatcaaa
     3121 gaccagcaag acaagatctg tgctgtgaag gccaacatcc tcaagaatga agaaaaaatc
     3181 cagaaaatgg tatatagtat caatttgact tcgagaaggt gactcgacgg taccgcgggc
     3241 ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     3301 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3361 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3421 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3481 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3541 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3601 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     3661 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     3721 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     3781 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     3841 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     3901 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     3961 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4021 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4081 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4141 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4201 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4261 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4321 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4381 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4441 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     4501 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4561 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4621 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     4681 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     4741 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     4801 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     4861 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     4921 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4981 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5041 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5101 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5161 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5221 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5281 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5341 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5401 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     5461 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5521 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     5581 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     5641 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     5701 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     5761 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5821 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5881 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5941 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6001 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6061 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6121 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6181 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6241 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6301 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6361 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6421 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6481 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6541 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
