LOCUS       sequence-337683-        5608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains mCherry-mNeonGreen tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5608)
  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 5608)
  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-mCherry-mNeonGreen-kanMX (pKBJ006)
FEATURES             Location/Qualifiers
     source          1..5608
                     /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..868
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(306..324)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     574..593
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1641..1828
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1696..1720)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1903..3259
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1903..2246
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2247..3056
                     /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(2314..2333)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3062..3259
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3363..3382)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3364..3382)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3469..3486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3640..4228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3720..3739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4399..5190,5191..5259))
                     /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     5022..5041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5260..5364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5432..5450
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5488..5510)
                     /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 agggcgagga
      181 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
      241 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
      301 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
      361 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
      421 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
      481 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
      541 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
      601 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
      661 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
      721 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
      781 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
      841 caccggcggc atggacgagc tgtacaagct cgacggagca ggtgctggtg ctggtgctgg
      901 agcaatggtt tctaagggtg aagaagacaa catggcttct ttgccagcta ctcacgaatt
      961 gcacatcttc ggttctatca acggtgttga cttcgacatg gttggtcaag gtactggtaa
     1021 cccaaacgac ggttacgaag aattgaactt gaagtctact aagggtgact tgcaattctc
     1081 tccatggatc ttggttccac acatcggtta cggtttccac caatacttgc catacccaga
     1141 cggtatgtct ccattccaag ctgctatggt tgacggttct ggttaccaag ttcacagaac
     1201 tatgcaattc gaagacggtg cttctttgac tgttaactac agatacactt acgaaggttc
     1261 tcacatcaag ggtgaagctc aagttaaggg tactggtttc ccagctgacg gtccagttat
     1321 gactaactct ttgactgctg ctgactggtg tagatctaag aagacttacc caaacgacaa
     1381 gactatcatc tctactttca agtggtctta cactactggt aacggtaaga gatacagatc
     1441 tactgctaga actacttaca ctttcgctaa gccaatggct gctaactact tgaagaacca
     1501 accaatgtac gttttcagaa agactgaatt gaagcactct aagactgaat tgaacttcaa
     1561 ggaatggcaa aaggctttca ctgacgttat gggtatggac gaattgtaca agggatccta
     1621 agcgcgccac ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag
     1681 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat
     1741 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg
     1801 tcgctcttat tgaccacacc tctaccggca gatccgctag ggataacagg gtaatataga
     1861 tctgtttagc ttgcctcgtc cccgccgggt cacccggcca gcgacatgga ggcccagaat
     1921 accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac
     1981 atttagccca tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg
     2041 gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa cgctcccctc
     2101 acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat
     2161 ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct
     2221 cacatcacat ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt tcgaggccgc
     2281 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     2341 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     2401 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     2461 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     2521 catggttact caccactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc
     2581 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     2641 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     2701 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     2761 ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg
     2821 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     2881 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     2941 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     3001 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     3061 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat
     3121 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat
     3181 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg
     3241 aatgctggtc gctatactgc tgtcgattcg atactaacgc cgccatccag tttaaacgag
     3301 ctcgaattca tcgatgatat cagatccact agtggcctat gcggccgcgg atctgccggt
     3361 ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg
     3421 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3481 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3541 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3601 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3661 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3721 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3781 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     3841 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3901 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3961 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4021 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4081 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4141 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4201 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4261 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4321 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4381 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4441 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4501 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4561 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4621 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4681 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4741 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4801 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4861 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4921 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4981 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5041 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5101 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5161 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5221 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5281 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5341 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     5401 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     5461 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     5521 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     5581 tggcgggtgt cggggctggc ttaactat
//
