LOCUS       sequence-254932-        4380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses iFAST in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4380)
  AUTHORS   Tebo AG, Pimenta FM, Zhang Y, Gautier A
  TITLE     Improved Chemical-Genetic Fluorescent Markers for Live Cell 
            Microscopy.
  JOURNAL   Biochemistry. 2018 Oct 2;57(39):5648-5653. doi: 
            10.1021/acs.biochem.8b00649. Epub 2018 Sep 17.
  PUBMED    30204425
REFERENCE   2  (bases 1 to 4380)
  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: 
            "10.1021/acs.biochem.8b00649"; journalName: "Biochemistry"; date: 
            "2018-10-2- 2"; volume: "57"; issue: "39"; pages: "5648-5653"
COMMENT     Sequence Label: pAG244 iFAST
FEATURES             Location/Qualifiers
     source          1..4380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        32..335
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        336..539
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     490..510
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      586..595
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             592..966
                     /codon_start=1
                     /product="small protein tag, derived from photoactive 
                     yellow protein, that allows in vivo fluorescent labeling by
                     addition of a cell-permeant fluorogen (Plamont et al., 
                     2016)"
                     /label=Y-FAST
                     /translation="MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTK
                     VKIHMKKALSGDSYWVFVKRV"
     CDS             973..1002
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1139..1260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1176..1195)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1230..1249
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1267..1722)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1404..1425)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1616..1635
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1749..1853
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1855..2212
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1855..2046
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1878..1898)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2063..2198
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2247..3041
                     /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             2247..3041
                     /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(2301..2320)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2911..2930
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3229..3248)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3273..3320
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3649..4237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4138..4157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
       61 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      121 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      181 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      241 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      301 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      361 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      421 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      481 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      541 gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctgccac catggagcat
      601 gttgcctttg gcagtgagga catcgagaac actctggcca aaatggacga cggacaactg
      661 gatgggttgg cctttggcgc aattcagctc gatggtgacg ggaatatcct gcagtacaat
      721 gctgctgaag gagacatcac aggcagagat cccaaacagg tgattgggaa gaacttcttc
      781 aaggatgttg cacctggaac ggattctccc gagttttacg gcaaattcaa ggaaggcgta
      841 gcgtcaggga atctgaacac catgttcgaa tggatgatac cgacaagcag gggaccaacc
      901 aaggtcaaga tacacatgaa gaaagccctt tccggtgaca gctattgggt ctttgtgaaa
      961 cgggtgggat ccgaacaaaa gcttatttct gaagaggact tgtaatagcc cgggtaagcg
     1021 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1081 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1141 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1201 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1261 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1321 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     1381 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     1441 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     1501 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1561 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     1621 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     1681 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     1741 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1801 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     1861 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     1921 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     1981 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2041 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2101 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     2161 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     2221 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2281 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2341 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2401 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     2461 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2521 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2581 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2641 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2701 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2761 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2821 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2881 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2941 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3001 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3061 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3121 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3181 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     3241 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     3301 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     3361 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     3421 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     3481 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3541 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3601 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3661 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3721 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3781 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3841 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3901 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3961 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4021 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4081 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4141 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4201 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4261 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4321 ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
//
