LOCUS       sequence-341935-        6673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-SWAT donor for Seamless tagging, contains mCherry-mNeonGreen 
            tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6673)
  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 6673)
  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 (pJJF003)
FEATURES             Location/Qualifiers
     source          1..6673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..66
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(104..126)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     226..245
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            259..1615
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        259..602
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             603..1412
                     /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(670..689)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1418..1615
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      complement(1705..2160)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1842..1863)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2054..2073
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2286..2308
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2300..2317
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2301..2317
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2327..2346
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2327..2345
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2427..3134
                     /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(2572..2590)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2840..2859
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3975..3995)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3975..3993)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4014..4030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4014..4030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4027..4049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4038..4054
                     /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(4062..4068,4069..4086,4087..4092))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4107..4128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4245..4262)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4416..5004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4496..4515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5175..5966,5967..6035))
                     /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     5798..5817
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6036..6140)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6177..6673,1..7)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 agcgatccgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
       61 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
      121 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
      181 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
      241 ttgtactgag agtgcagcga catggaggcc cagaataccc tccttgacag tcttgacgtg
      301 cgcagctcag gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta
      361 taatcatttg catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc
      421 tcgctgcaga cctgcgagca gggaaacgct cccctcacag acgcgttgaa ttgtccccac
      481 gccgcgcccc tgtagagaaa tataaaaggt taggatttgc cactgaggtt cttctttcat
      541 atacttcctt ttaaaatctt gctaggatac agttctcaca tcacatccga acataaacaa
      601 ccatgggtaa ggaaaagact cacgtttcga ggccgcgatt aaattccaac atggatgctg
      661 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
      721 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
      781 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
      841 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
      901 ccggcaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
      961 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     1021 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     1081 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     1141 tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     1201 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     1261 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     1321 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     1381 agtttcattt gatgctcgat gagtttttct aatcagtact gacaataaaa agattcttgt
     1441 tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa
     1501 tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag
     1561 tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc
     1621 gattcgatac taacgccgcc atccagtgtc gaaaacgcgt gtgaaatacc gcacagatgc
     1681 gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
     1741 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1801 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1861 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1921 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     1981 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2041 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2101 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2161 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     2221 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     2281 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     2341 atagggcgaa ttgggtaccc tatattaccc tgttatccct acgtacgctg caggtcgacg
     2401 gtggcggttc tggcggtggc ggatccatgg tgagcaaggg cgaggaggat aacatggcca
     2461 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
     2521 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     2581 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
     2641 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     2701 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     2761 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     2821 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     2881 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     2941 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
     3001 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
     3061 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     3121 acgagctgta caagctcgac ggagcaggtg ctggtgctgg tgctggagca atggtttcta
     3181 agggtgaaga agacaacatg gcttctttgc cagctactca cgaattgcac atcttcggtt
     3241 ctatcaacgg tgttgacttc gacatggttg gtcaaggtac tggtaaccca aacgacggtt
     3301 acgaagaatt gaacttgaag tctactaagg gtgacttgca attctctcca tggatcttgg
     3361 ttccacacat cggttacggt ttccaccaat acttgccata cccagacggt atgtctccat
     3421 tccaagctgc tatggttgac ggttctggtt accaagttca cagaactatg caattcgaag
     3481 acggtgcttc tttgactgtt aactacagat acacttacga aggttctcac atcaagggtg
     3541 aagctcaagt taagggtact ggtttcccag ctgacggtcc agttatgact aactctttga
     3601 ctgctgctga ctggtgtaga tctaagaaga cttacccaaa cgacaagact atcatctcta
     3661 ctttcaagtg gtcttacact actggtaacg gtaagagata cagatctact gctagaacta
     3721 cttacacttt cgctaagcca atggctgcta actacttgaa gaaccaacca atgtacgttt
     3781 tcagaaagac tgaattgaag cactctaaga ctgaattgaa cttcaaggaa tggcaaaagg
     3841 ctttcactga cgttatgggt atggacgaat tgtacaagta aactagtagt tcttctttga
     3901 gatatcgatt gaacgagctc gaattcatcg atctatatta ccctgttatc cctagcgtaa
     3961 ctcagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     4021 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4081 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4141 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4201 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4261 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4321 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4381 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4441 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4501 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4561 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4621 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4681 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4741 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4801 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4861 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4921 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4981 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5041 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5101 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5161 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5221 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5281 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5341 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5401 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5461 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5521 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5581 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5641 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5701 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5761 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5821 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5881 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5941 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6001 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6061 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6121 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgggtc cttttcatca
     6181 cgtgctataa aaataattat aatttaaatt ttttaatata aatatataaa ttaaaaatag
     6241 aaagtaaaaa aagaaattaa agaaaaaata gtttttgttt tccgaagatg taaaagactc
     6301 tagggggatc gccaacaaat actacctttt atcttgctct tcctgctctc aggtattaat
     6361 gccgaattgt ttcatcttgt ctgtgtagaa gaccacacac gaaaatcctg tgattttaca
     6421 ttttacttat cgttaatcga atgtatatct atttaatctg cttttcttgt ctaataaata
     6481 tatatgtaaa gtacgctttt tgttgaaatt ttttaaacct ttgtttattt ttttttcttc
     6541 attccgtaac tcttctacct tctttattta ctttctaaaa tccaaataca aaacataaaa
     6601 ataaataaac acagagtaaa ttcccaaatt attccatcat taaaagatac gaggcgcgtg
     6661 taagttacag gca
//
