LOCUS       sequence-341933-        8160 bp    DNA     circular SYN 15-APR-2025
DEFINITION  C-SWAT donor for Selection Reconstitution tagging, contains 
            mCherry-sfGFP tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8160)
  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 8160)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pRS41K-mCherry-sfGFP-ADH1term-TEFpr-hph-Delta-C 
            (pJJ...
FEATURES             Location/Qualifiers
     source          1..8160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..40)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     140..159
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            173..1529
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        173..516
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             517..1326
                     /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(584..603)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1332..1529
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1619..2074)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1756..1777)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1968..1987
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2200..2222
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2214..2231
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2215..2231
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2241..2260
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2241..2259
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2341..3048
                     /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(2486..2504)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2754..2773
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3821..4008
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3876..3900)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        4056..4399
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(5376..5396)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5376..5394)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5415..5431)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5415..5431)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5428..5450)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5439..5455
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5463..5469,5470..5487,5488..5493))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5508..5529
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5646..5663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5817..6405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5897..5916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6576..7367,7368..7436))
                     /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     7199..7218
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7437..7541)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7578..8081
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     8122..8140
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
       61 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
      121 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca gcgacatgga
      181 ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg
      241 tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga
      301 tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa
      361 cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa
      421 aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg
      481 atacagttct cacatcacat ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt
      541 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
      601 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
      661 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
      721 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
      781 cctgatgatg catggttact caccactgcg atccccggca aaacagcatt ccaggtatta
      841 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
      901 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
      961 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     1021 aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg
     1081 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     1141 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     1201 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     1261 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     1321 ttctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt
     1381 ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc
     1441 gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca
     1501 atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc cgccatccag
     1561 tgtcgaaaac gcgtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     1621 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     1681 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     1741 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1801 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     1861 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     1921 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     1981 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     2041 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     2101 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     2161 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     2221 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt accctatatt
     2281 accctgttat ccctacgtac gctgcaggtc gacggtggcg gttctggcgg tggcggatcc
     2341 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     2401 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     2461 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     2521 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     2581 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     2641 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     2701 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     2761 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     2821 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     2881 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     2941 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     3001 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagct cgacggagca
     3061 ggtgctggtg ctggtgctgg agcaatgtcc aagggtgaag agctatttac tggggttgta
     3121 cccattttgg tagaactgga cggagatgta aacggacata aattctctgt tagaggtgag
     3181 ggcgaaggcg atgccaccaa tggtaaattg actctgaagt ttatatgcac tacgggtaaa
     3241 ttacctgttc cttggccaac cctagtaaca actttgacat atggtgttca atgtttctca
     3301 agatacccag accatatgaa aaggcatgat ttctttaaaa gtgctatgcc agaaggctac
     3361 gtgcaagaga gaactatctc ctttaaggat gacggtacgt ataaaacacg agcagaagtg
     3421 aaattcgaag gggatacact agttaatcgc atcgaattaa agggtataga ctttaaggaa
     3481 gatggtaata ttctcggcca taaacttgag tataatttca actcgcataa tgtgtacatt
     3541 acagctgaca aacaaaagaa cggaattaaa gcgaatttta aaatcaggca caacgtcgaa
     3601 gatgggtctg ttcaacttgc cgatcattat cagcaaaaca cccctattgg tgatggtcca
     3661 gtcttgttac ccgataatca ctacttaagc acacagtcta gattgtcaaa agatccgaat
     3721 gaaaagcgtg atcacatggt tttattggaa tttgtcaccg ctgcaggaat aactcacgga
     3781 atggacgagc tttataagta aactagtcac ttctaaataa gcgaatttct tatgatttat
     3841 gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc
     3901 ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct
     3961 ttctcaggta tagtatgagg tcgctcttat tgaccacacc tctaccggca agatctgttt
     4021 agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
     4081 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
     4141 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
     4201 gcaaaaatta cggctcctcg ctgcggacct gcgagcaggg aaacgctccc ctcacagacg
     4261 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
     4321 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
     4381 catccgaaca taaacaacca tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     4441 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     4501 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     4561 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     4621 gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg
     4681 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     4741 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     4801 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     4861 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     4921 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtcctata
     4981 ttaccctgtt atccctagcg taacttaatt ggtacactaa tcaattaaag agcacttctc
     5041 tggttggaat atccattgct atcaaaatag tagattcccg cctattaata ttcttatgcc
     5101 caagagttgg gaattgaaag gaaataaaaa atcaaataaa aaatcaatca tcatgcacca
     5161 gaaatagaaa aatacatata tacttaagca acactaagat tgcaacttga aaccaagttt
     5221 tggtaaaaat taaatgcaag atttacaaat atgaggtgta gggagtgtat actatatgca
     5281 gatatcaaat agttaaaaac taatgaaatg taaactagaa atatggtgtt cggaactttt
     5341 caatataatt aggaacgaca cggcagcttt tgttcccttt agtgagggtt aattgcgcgc
     5401 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5461 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5521 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5581 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5641 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5701 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5761 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5821 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5881 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5941 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6001 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6061 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6121 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6181 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6241 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6301 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6361 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6421 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6481 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6541 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6601 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6661 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6721 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6781 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6841 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6901 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6961 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7021 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7081 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7141 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7201 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7261 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7321 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7381 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7441 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7501 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7561 ccacctgggt ccttttcatc acgtgctata aaaataatta taatttaaat tttttaatat
     7621 aaatatataa attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt
     7681 ttccgaagat gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc
     7741 ttcctgctct caggtattaa tgccgaattg tttcatcttg tctgtgtaga agaccacaca
     7801 cgaaaatcct gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct
     7861 gcttttcttg tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc
     7921 tttgtttatt tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa
     7981 atccaaatac aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca
     8041 ttaaaagata cgaggcgcgt gtaagttaca ggcaagcgat ccgtctaaga aaccattatt
     8101 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
//
