LOCUS       sequence-341929-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-SWAT donor for Seamless tagging, contains mCherry-sfGFP tandem 
            fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  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 6679)
  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-sfGFP (pJJF001)
FEATURES             Location/Qualifiers
     source          1..6679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..80
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(118..140)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     240..259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            273..1629
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        273..616
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             617..1426
                     /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(684..703)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1432..1629
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1719..2174)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1856..1877)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2068..2087
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2300..2322
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2314..2331
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2315..2331
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2341..2360
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2341..2359
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2441..3148
                     /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(2586..2604)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2854..2873
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3995..4015)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3995..4013)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4034..4050)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4034..4050)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4047..4069)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4058..4074
                     /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(4082..4088,4089..4106,4107..4112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4127..4148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4265..4282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4436..5024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4516..4535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5195..5986,5987..6055))
                     /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     5818..5837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6056..6160)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6197..6679,1..21)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 gtaagttaca ggcaagcgat ccgtctaaga aaccattatt atcatgacat taacctataa
       61 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
      121 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
      181 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
      241 ggcatcagag cagattgtac tgagagtgca gcgacatgga ggcccagaat accctccttg
      301 acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac atttagccca
      361 tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg gcgcgaagca
      421 aaaattacgg ctcctcgctg cagacctgcg agcagggaaa cgctcccctc acagacgcgt
      481 tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat ttgccactga
      541 ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct cacatcacat
      601 ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt tcgaggccgc gattaaattc
      661 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
      721 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
      781 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
      841 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
      901 caccactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc ctgattcagg
      961 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     1021 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     1081 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     1141 agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg tcactcatgg
     1201 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     1261 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     1321 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     1381 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag tactgacaat
     1441 aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt
     1501 ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca
     1561 gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc
     1621 gctatactgc tgtcgattcg atactaacgc cgccatccag tgtcgaaaac gcgtgtgaaa
     1681 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt
     1741 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     1801 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     1861 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     1921 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     1981 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2041 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2101 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2161 gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     2221 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     2281 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     2341 taatacgact cactataggg cgaattgggt accctatatt accctgttat ccctacgtac
     2401 gctgcaggtc gacggtggcg gttctggcgg tggcggatcc atggtgagca agggcgagga
     2461 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     2521 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     2581 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     2641 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     2701 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     2761 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     2821 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     2881 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     2941 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     3001 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     3061 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     3121 caccggcggc atggacgagc tgtacaagct cgacggagca ggtgctggtg ctggtgctgg
     3181 agcaatgtcc aagggtgaag agctatttac tggggttgta cccattttgg tagaactgga
     3241 cggagatgta aacggacata aattctctgt tagaggtgag ggcgaaggcg atgccaccaa
     3301 tggtaaattg actctgaagt ttatatgcac tacgggtaaa ttacctgttc cttggccaac
     3361 cctagtaaca actttgacat atggtgttca atgtttctca agatacccag accatatgaa
     3421 aaggcatgat ttctttaaaa gtgctatgcc agaaggctac gtgcaagaga gaactatctc
     3481 ctttaaggat gacggtacgt ataaaacacg agcagaagtg aaattcgaag gggatacact
     3541 agttaatcgc atcgaattaa agggtataga ctttaaggaa gatggtaata ttctcggcca
     3601 taaacttgag tataatttca actcgcataa tgtgtacatt acagctgaca aacaaaagaa
     3661 cggaattaaa gcgaatttta aaatcaggca caacgtcgaa gatgggtctg ttcaacttgc
     3721 cgatcattat cagcaaaaca cccctattgg tgatggtcca gtcttgttac ccgataatca
     3781 ctacttaagc acacagtcta gattgtcaaa agatccgaat gaaaagcgtg atcacatggt
     3841 tttattggaa tttgtcaccg ctgcaggaat aactcacgga atggacgagc tttataagta
     3901 aactagtagt tcttctttga gatatcgatt gaacgagctc gaattcatcg atctatatta
     3961 ccctgttatc cctagcgtaa ctcagctttt gttcccttta gtgagggtta attgcgcgct
     4021 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4081 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4141 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4201 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4261 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4321 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4441 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4621 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4861 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5041 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5101 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5161 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5221 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5281 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5341 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5401 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5461 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5521 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5581 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5641 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5701 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5761 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5821 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5881 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5941 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6001 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6061 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6121 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6181 cacctgggtc cttttcatca cgtgctataa aaataattat aatttaaatt ttttaatata
     6241 aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa agaaaaaata gtttttgttt
     6301 tccgaagatg taaaagactc tagggggatc gccaacaaat actacctttt atcttgctct
     6361 tcctgctctc aggtattaat gccgaattgt ttcatcttgt ctgtgtagaa gaccacacac
     6421 gaaaatcctg tgattttaca ttttacttat cgttaatcga atgtatatct atttaatctg
     6481 cttttcttgt ctaataaata tatatgtaaa gtacgctttt tgttgaaatt ttttaaacct
     6541 ttgtttattt ttttttcttc attccgtaac tcttctacct tctttattta ctttctaaaa
     6601 tccaaataca aaacataaaa ataaataaac acagagtaaa ttcccaaatt attccatcat
     6661 taaaagatac gaggcgcgt
//
