LOCUS       sequence-250340-        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        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      615..624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             621..995
                     /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             1002..1031
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1168..1289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1205..1224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1259..1278
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1296..1751)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1433..1454)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1645..1664
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1778..1882
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1884..2241
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1884..2075
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1907..1927)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2092..2227
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2276..3070
                     /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             2276..3070
                     /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(2330..2349)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2940..2959
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3258..3277)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3302..3349
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3678..4266
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4167..4186
                     /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 ccggactcag atctgccacc atggagcatg ttgcctttgg cagtgaggac atcgagaaca
      661 ctctggccaa aatggacgac ggacaactgg atgggttggc ctttggcgca attcagctcg
      721 atggtgacgg gaatatcctg cagtacaatg ctgctgaagg agacatcaca ggcagagatc
      781 ccaaacaggt gattgggaag aacttcttca aggatgttgc acctggaacg gattctcccg
      841 agttttacgg caaattcaag gaaggcgtag cgtcagggaa tctgaacacc atgttcgaat
      901 ggatgatacc gacaagcagg ggaccaacca aggtcaagat acacatgaag aaagcccttt
      961 ccggtgacag ctattgggtc tttgtgaaac gggtgggatc cgaacaaaag cttatttctg
     1021 aagaggactt gtaatagccc gggtaagcgg ccgcgactct agatcataat cagccatacc
     1081 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     1141 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     1201 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     1261 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     1321 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     1381 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     1441 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     1501 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     1561 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     1621 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     1681 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     1741 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1801 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1861 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     1921 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1981 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2041 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2101 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2161 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     2221 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     2281 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     2341 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     2401 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     2461 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     2521 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     2581 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     2641 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     2701 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     2761 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     2821 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     2881 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     2941 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     3001 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     3061 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     3121 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     3181 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     3241 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     3301 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     3361 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     3421 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     3481 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     3541 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3601 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3661 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3721 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3781 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3841 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3901 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3961 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4021 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4081 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4141 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4201 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4261 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4321 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
