LOCUS       sequence-341941-        8142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-SWAT donor for Selection Reconstitution tagging, contains 
            mScarletI-mNeonGreen tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8142)
  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 8142)
  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: pRS41K-mScarlet-I-mNeonGreen-ADH1term-TEFpr-hp...
FEATURES             Location/Qualifiers
     source          1..8142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..61
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(99..121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     221..240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            254..1610
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        254..597
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             598..1407
                     /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(665..684)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1413..1610
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1700..2155)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1837..1858)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2049..2068
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2281..2303
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2295..2312
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2296..2312
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2322..2341
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2322..2340
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2422..3117
                     /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(2555..2573)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2823..2842
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3884..4071
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3939..3963)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        4119..4462
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(5439..5459)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5439..5457)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5478..5494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5478..5494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5491..5513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5502..5518
                     /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(5526..5532,5533..5550,5551..5556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5571..5592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5709..5726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5880..6468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5960..5979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6639..7430,7431..7499))
                     /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     7262..7281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7500..7604)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(7641..8142,1..2)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 tccgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
       61 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      121 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      181 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      241 ctgagagtgc agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag
      301 ctcaggggca tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc
      361 atttgcatcc atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct
      421 gcagacctgc gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc
      481 gcccctgtag agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact
      541 tccttttaaa atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg
      601 ggtaaggaaa agactcacgt ttcgaggccg cgattaaatt ccaacatgga tgctgattta
      661 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
      721 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
      781 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
      841 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggc
      901 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
      961 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     1021 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     1081 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     1141 aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     1201 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     1261 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     1321 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     1381 catttgatgc tcgatgagtt tttctaatca gtactgacaa taaaaagatt cttgttttca
     1441 agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta
     1501 gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc
     1561 agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc
     1621 gatactaacg ccgccatcca gtgtcgaaaa cgcgtgtgaa ataccgcaca gatgcgtaag
     1681 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1741 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1801 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1861 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1921 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     1981 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2041 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2101 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt
     2161 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     2221 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     2281 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
     2341 gcgaattggg taccctatat taccctgtta tccctacgta cgctgcaggt cgacggtggc
     2401 ggttctggcg gtggcggatc catggtgagc aagggcgagg cagtgatcaa ggagttcatg
     2461 cggttcaagg tgcacatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     2521 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     2581 cccctgccct tctcctggga catcctgtcc cctcagttca tgtacggctc cagggccttc
     2641 atcaagcacc ccgccgacat ccccgactac tataagcagt ccttccccga gggcttcaag
     2701 tgggagcgcg tgatgaactt cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc
     2761 ctggaggacg gcaccctgat ctacaaggtg aagctccgcg gcaccaactt ccctcctgac
     2821 ggccccgtaa tgcagaagaa gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc
     2881 gaggacggcg tgctgaaggg cgacattaag atggccctgc gcctgaagga cggcggccgc
     2941 tacctggcgg acttcaagac cacctacaag gccaagaagc ccgtgcagat gcccggcgcc
     3001 tacaacgtcg accgcaagtt ggacatcacc tcccacaacg aggactacac cgtggtggaa
     3061 cagtacgaac gctccgaggg ccgccactcc accggcggca tggacgagct gtacaagctc
     3121 gacggagcag gtgctggtgc tggtgctgga gcaatggttt ctaagggtga agaagacaac
     3181 atggcttctt tgccagctac tcacgaattg cacatcttcg gttctatcaa cggtgttgac
     3241 ttcgacatgg ttggtcaagg tactggtaac ccaaacgacg gttacgaaga attgaacttg
     3301 aagtctacta agggtgactt gcaattctct ccatggatct tggttccaca catcggttac
     3361 ggtttccacc aatacttgcc atacccagac ggtatgtctc cattccaagc tgctatggtt
     3421 gacggttctg gttaccaagt tcacagaact atgcaattcg aagacggtgc ttctttgact
     3481 gttaactaca gatacactta cgaaggttct cacatcaagg gtgaagctca agttaagggt
     3541 actggtttcc cagctgacgg tccagttatg actaactctt tgactgctgc tgactggtgt
     3601 agatctaaga agacttaccc aaacgacaag actatcatct ctactttcaa gtggtcttac
     3661 actactggta acggtaagag atacagatct actgctagaa ctacttacac tttcgctaag
     3721 ccaatggctg ctaactactt gaagaaccaa ccaatgtacg ttttcagaaa gactgaattg
     3781 aagcactcta agactgaatt gaacttcaag gaatggcaaa aggctttcac tgacgttatg
     3841 ggtatggacg aattgtacaa gtaaactagt cacttctaaa taagcgaatt tcttatgatt
     3901 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga
     3961 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt
     4021 gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg gcaagatctg
     4081 tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc cagaataccc
     4141 tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt
     4201 agcccataca tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc
     4261 gaagcaaaaa ttacggctcc tcgctgcgga cctgcgagca gggaaacgct cccctcacag
     4321 acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc
     4381 cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca
     4441 tcacatccga acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga
     4501 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     4561 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     4621 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     4681 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     4741 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     4801 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     4861 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     4921 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     4981 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtcct
     5041 atattaccct gttatcccta gcgtaactta attggtacac taatcaatta aagagcactt
     5101 ctctggttgg aatatccatt gctatcaaaa tagtagattc ccgcctatta atattcttat
     5161 gcccaagagt tgggaattga aaggaaataa aaaatcaaat aaaaaatcaa tcatcatgca
     5221 ccagaaatag aaaaatacat atatacttaa gcaacactaa gattgcaact tgaaaccaag
     5281 ttttggtaaa aattaaatgc aagatttaca aatatgaggt gtagggagtg tatactatat
     5341 gcagatatca aatagttaaa aactaatgaa atgtaaacta gaaatatggt gttcggaact
     5401 tttcaatata attaggaacg acacggcagc ttttgttccc tttagtgagg gttaattgcg
     5461 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5521 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5581 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5641 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5701 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5761 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5821 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5881 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5941 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6001 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6061 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6121 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6181 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6241 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6301 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6361 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6421 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6481 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6541 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6601 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6661 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6721 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6781 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6841 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6901 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6961 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7021 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7081 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7141 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7201 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7261 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7321 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7381 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7441 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7501 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7561 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7621 gtgccacctg ggtccttttc atcacgtgct ataaaaataa ttataattta aattttttaa
     7681 tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt
     7741 gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc ttttatcttg
     7801 ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac
     7861 acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat atctatttaa
     7921 tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga aattttttaa
     7981 acctttgttt attttttttt cttcattccg taactcttct accttcttta tttactttct
     8041 aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca aattattcca
     8101 tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc ga
//
