LOCUS       sequence-341939-        8154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-SWAT donor for Selection Reconstitution tagging, contains 
            mCherry-mNeonGreen tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8154)
  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 8154)
  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-mCherry-mNeonGreen-ADH1term-TEFpr-hph-Delta-C...
FEATURES             Location/Qualifiers
     source          1..8154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..21
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(59..81)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     181..200
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            214..1570
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        214..557
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             558..1367
                     /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(625..644)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1373..1570
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1660..2115)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1797..1818)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2009..2028
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2241..2263
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2255..2272
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2256..2272
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2282..2301
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2282..2300
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2382..3089
                     /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(2527..2545)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2795..2814
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3856..4043
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3911..3935)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        4091..4434
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(5411..5431)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5411..5429)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5450..5466)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5450..5466)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5463..5485)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5474..5490
                     /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(5498..5504,5505..5522,5523..5528))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5543..5564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5681..5698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5852..6440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5932..5951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6611..7402,7403..7471))
                     /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     7234..7253
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7472..7576)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7613..8116
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
       61 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
      121 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
      181 cggcatcaga gcagattgta ctgagagtgc agcgacatgg aggcccagaa taccctcctt
      241 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
      301 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
      361 aaaaattacg gctcctcgct gcagacctgc gagcagggaa acgctcccct cacagacgcg
      421 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
      481 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
      541 tccgaacata aacaaccatg ggtaaggaaa agactcacgt ttcgaggccg cgattaaatt
      601 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
      661 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
      721 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
      781 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
      841 tcaccactgc gatccccggc aaaacagcat tccaggtatt agaagaatat cctgattcag
      901 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
      961 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     1021 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     1081 aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg
     1141 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     1201 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     1261 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     1321 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gtactgacaa
     1381 taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt
     1441 tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc
     1501 agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt
     1561 cgctatactg ctgtcgattc gatactaacg ccgccatcca gtgtcgaaaa cgcgtgtgaa
     1621 ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt
     1681 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1741 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1801 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     1861 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     1921 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     1981 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2041 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2101 cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     2161 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
     2221 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc
     2281 gtaatacgac tcactatagg gcgaattggg taccctatat taccctgtta tccctacgta
     2341 cgctgcaggt cgacggtggc ggttctggcg gtggcggatc catggtgagc aagggcgagg
     2401 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     2461 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     2521 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     2581 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     2641 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     2701 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     2761 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     2821 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     2881 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     2941 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     3001 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     3061 ccaccggcgg catggacgag ctgtacaagc tcgacggagc aggtgctggt gctggtgctg
     3121 gagcaatggt ttctaagggt gaagaagaca acatggcttc tttgccagct actcacgaat
     3181 tgcacatctt cggttctatc aacggtgttg acttcgacat ggttggtcaa ggtactggta
     3241 acccaaacga cggttacgaa gaattgaact tgaagtctac taagggtgac ttgcaattct
     3301 ctccatggat cttggttcca cacatcggtt acggtttcca ccaatacttg ccatacccag
     3361 acggtatgtc tccattccaa gctgctatgg ttgacggttc tggttaccaa gttcacagaa
     3421 ctatgcaatt cgaagacggt gcttctttga ctgttaacta cagatacact tacgaaggtt
     3481 ctcacatcaa gggtgaagct caagttaagg gtactggttt cccagctgac ggtccagtta
     3541 tgactaactc tttgactgct gctgactggt gtagatctaa gaagacttac ccaaacgaca
     3601 agactatcat ctctactttc aagtggtctt acactactgg taacggtaag agatacagat
     3661 ctactgctag aactacttac actttcgcta agccaatggc tgctaactac ttgaagaacc
     3721 aaccaatgta cgttttcaga aagactgaat tgaagcactc taagactgaa ttgaacttca
     3781 aggaatggca aaaggctttc actgacgtta tgggtatgga cgaattgtac aagtaaacta
     3841 gtcacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata
     3901 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     3961 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta tgaggtcgct
     4021 cttattgacc acacctctac cggcaagatc tgtttagctt gcctcgtccc cgccgggtca
     4081 cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc
     4141 aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt
     4201 tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgcg
     4261 gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc
     4321 cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc
     4381 ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt
     4441 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     4501 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     4561 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     4621 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     4681 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     4741 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     4801 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     4861 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     4921 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     4981 atgctttggg ccgaggactg ccccgaagtc ctatattacc ctgttatccc tagcgtaact
     5041 taattggtac actaatcaat taaagagcac ttctctggtt ggaatatcca ttgctatcaa
     5101 aatagtagat tcccgcctat taatattctt atgcccaaga gttgggaatt gaaaggaaat
     5161 aaaaaatcaa ataaaaaatc aatcatcatg caccagaaat agaaaaatac atatatactt
     5221 aagcaacact aagattgcaa cttgaaacca agttttggta aaaattaaat gcaagattta
     5281 caaatatgag gtgtagggag tgtatactat atgcagatat caaatagtta aaaactaatg
     5341 aaatgtaaac tagaaatatg gtgttcggaa cttttcaata taattaggaa cgacacggca
     5401 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     5461 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5521 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5581 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5641 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5701 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5761 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5821 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5881 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5941 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6001 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6061 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6121 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6181 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6241 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6301 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6361 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6421 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6481 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6541 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6601 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6661 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6721 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6781 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6841 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6901 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6961 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7021 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7081 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7141 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7201 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7261 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7321 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7381 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7441 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7501 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7561 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgggtccttt tcatcacgtg
     7621 ctataaaaat aattataatt taaatttttt aatataaata tataaattaa aaatagaaag
     7681 taaaaaaaga aattaaagaa aaaatagttt ttgttttccg aagatgtaaa agactctagg
     7741 gggatcgcca acaaatacta ccttttatct tgctcttcct gctctcaggt attaatgccg
     7801 aattgtttca tcttgtctgt gtagaagacc acacacgaaa atcctgtgat tttacatttt
     7861 acttatcgtt aatcgaatgt atatctattt aatctgcttt tcttgtctaa taaatatata
     7921 tgtaaagtac gctttttgtt gaaatttttt aaacctttgt ttattttttt ttcttcattc
     7981 cgtaactctt ctaccttctt tatttacttt ctaaaatcca aatacaaaac ataaaaataa
     8041 ataaacacag agtaaattcc caaattattc catcattaaa agatacgagg cgcgtgtaag
     8101 ttacaggcaa gcgatccgtc taagaaacca ttattatcat gacattaacc tata
//
