LOCUS       sequence-341921-        5596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains mScarletI-mNeonGreen tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5596)
  AUTHORS   Fung JJ, Blocher-Juarez K, Khmelinskii A
  TITLE     High-Throughput Analysis of Protein Turnover with Tandem Fluorescent
            Protein Timers.
  JOURNAL   Methods Mol Biol. 2022;2378:85-100. doi: 
            10.1007/978-1-0716-1732-8_6.
  PUBMED    34985695
REFERENCE   2  (bases 1 to 5596)
  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: "Methods Mol
            Biol."; date: "2022"; volume: "2378:85-100. doi"; pages: " 
            10.1007/978-1-0716-1732-8_6"
COMMENT     Sequence Label: pFA6a-mScarlet-I-mNeonGreen-kanMX (pKBJ007)
FEATURES             Location/Qualifiers
     source          1..5596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        102..120
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     102..119
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             161..856
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(294..312)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     562..581
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1629..1816
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1684..1708)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1891..3247
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1891..2234
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2235..3044
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2302..2321)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3050..3247
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3351..3370)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3352..3370)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3457..3474)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3628..4216)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3708..3727)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4387..5178,5179..5247))
                     /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     5010..5029
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5248..5352)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5420..5438
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5476..5498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc
       61 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactataga
      121 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac atggtgagca agggcgaggc
      181 agtgatcaag gagttcatgc ggttcaaggt gcacatggag ggctccatga acggccacga
      241 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
      301 gaaggtgacc aagggtggcc ccctgccctt ctcctgggac atcctgtccc ctcagttcat
      361 gtacggctcc agggccttca tcaagcaccc cgccgacatc cccgactact ataagcagtc
      421 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac
      481 cgtgacccag gacacctccc tggaggacgg caccctgatc tacaaggtga agctccgcgg
      541 caccaacttc cctcctgacg gccccgtaat gcagaagaag acaatgggct gggaagcgtc
      601 caccgagcgg ttgtaccccg aggacggcgt gctgaagggc gacattaaga tggccctgcg
      661 cctgaaggac ggcggccgct acctggcgga cttcaagacc acctacaagg ccaagaagcc
      721 cgtgcagatg cccggcgcct acaacgtcga ccgcaagttg gacatcacct cccacaacga
      781 ggactacacc gtggtggaac agtacgaacg ctccgagggc cgccactcca ccggcggcat
      841 ggacgagctg tacaagctcg acggagcagg tgctggtgct ggtgctggag caatggtttc
      901 taagggtgaa gaagacaaca tggcttcttt gccagctact cacgaattgc acatcttcgg
      961 ttctatcaac ggtgttgact tcgacatggt tggtcaaggt actggtaacc caaacgacgg
     1021 ttacgaagaa ttgaacttga agtctactaa gggtgacttg caattctctc catggatctt
     1081 ggttccacac atcggttacg gtttccacca atacttgcca tacccagacg gtatgtctcc
     1141 attccaagct gctatggttg acggttctgg ttaccaagtt cacagaacta tgcaattcga
     1201 agacggtgct tctttgactg ttaactacag atacacttac gaaggttctc acatcaaggg
     1261 tgaagctcaa gttaagggta ctggtttccc agctgacggt ccagttatga ctaactcttt
     1321 gactgctgct gactggtgta gatctaagaa gacttaccca aacgacaaga ctatcatctc
     1381 tactttcaag tggtcttaca ctactggtaa cggtaagaga tacagatcta ctgctagaac
     1441 tacttacact ttcgctaagc caatggctgc taactacttg aagaaccaac caatgtacgt
     1501 tttcagaaag actgaattga agcactctaa gactgaattg aacttcaagg aatggcaaaa
     1561 ggctttcact gacgttatgg gtatggacga attgtacaag ggatcctaag cgcgccactt
     1621 ctaaataagc gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa
     1681 taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc ttattcttga
     1741 gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gtatgaggtc gctcttattg
     1801 accacacctc taccggcaga tccgctaggg ataacagggt aatatagatc tgtttagctt
     1861 gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac
     1921 agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata
     1981 catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa
     2041 aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac agacgcgttg
     2101 aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg
     2161 ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc
     2221 gaacataaac aaccatgggt aaggaaaaga ctcacgtttc gaggccgcga ttaaattcca
     2281 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     2341 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     2401 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     2461 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     2521 ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     2581 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     2641 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     2701 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     2761 tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2821 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     2881 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     2941 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     3001 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagta ctgacaataa
     3061 aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg tagttgttct
     3121 attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca tctgcccaga
     3181 tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc
     3241 tatactgctg tcgattcgat actaacgccg ccatccagtt taaacgagct cgaattcatc
     3301 gatgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct ccctatagtg
     3361 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg
     3421 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3481 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3541 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3601 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3661 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3721 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3781 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3841 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3901 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3961 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4021 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4081 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4141 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4201 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4261 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4321 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4381 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4441 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4501 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4561 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4621 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4681 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4741 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4801 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4861 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4921 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4981 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5041 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5101 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5161 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5221 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5281 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5341 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5401 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     5461 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5521 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5581 gggctggctt aactat
//
