LOCUS       sequence-341940-        6661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-SWAT donor for Seamless tagging, contains mScarletI-mNeonGreen 
            tandem fluorescent protein timer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6661)
  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 6661)
  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 (pKBJ010)
FEATURES             Location/Qualifiers
     source          1..6661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(81..536)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(218..239)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     430..449
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     662..684
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     676..693
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     677..693
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     703..722
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        703..721
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             803..1498
                     /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(936..954)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1204..1223
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2339..2359)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2339..2357)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2378..2394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2378..2394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2391..2413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2402..2418
                     /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(2426..2432,2433..2450,2451..2456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2471..2492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2609..2626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2780..3368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2860..2879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3539..4330,4331..4399))
                     /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     4162..4181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4400..4504)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    4541..5044
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5085..5103
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5141..5163)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5263..5282
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            5296..6652
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        5296..5639
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             5640..6449
                     /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(5707..5726)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      6455..6652
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 cgatactaac gccgccatcc agtgtcgaaa acgcgtgtga aataccgcac agatgcgtaa
       61 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
      121 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
      181 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
      241 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
      301 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
      361 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
      421 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
      481 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat
      541 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
      601 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
      661 tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag
      721 ggcgaattgg gtaccctata ttaccctgtt atccctacgt acgctgcagg tcgacggtgg
      781 cggttctggc ggtggcggat ccatggtgag caagggcgag gcagtgatca aggagttcat
      841 gcggttcaag gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga
      901 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      961 ccccctgccc ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt
     1021 catcaagcac cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa
     1081 gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc
     1141 cctggaggac ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga
     1201 cggccccgta atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc
     1261 cgaggacggc gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg
     1321 ctacctggcg gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc
     1381 ctacaacgtc gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga
     1441 acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagct
     1501 cgacggagca ggtgctggtg ctggtgctgg agcaatggtt tctaagggtg aagaagacaa
     1561 catggcttct ttgccagcta ctcacgaatt gcacatcttc ggttctatca acggtgttga
     1621 cttcgacatg gttggtcaag gtactggtaa cccaaacgac ggttacgaag aattgaactt
     1681 gaagtctact aagggtgact tgcaattctc tccatggatc ttggttccac acatcggtta
     1741 cggtttccac caatacttgc catacccaga cggtatgtct ccattccaag ctgctatggt
     1801 tgacggttct ggttaccaag ttcacagaac tatgcaattc gaagacggtg cttctttgac
     1861 tgttaactac agatacactt acgaaggttc tcacatcaag ggtgaagctc aagttaaggg
     1921 tactggtttc ccagctgacg gtccagttat gactaactct ttgactgctg ctgactggtg
     1981 tagatctaag aagacttacc caaacgacaa gactatcatc tctactttca agtggtctta
     2041 cactactggt aacggtaaga gatacagatc tactgctaga actacttaca ctttcgctaa
     2101 gccaatggct gctaactact tgaagaacca accaatgtac gttttcagaa agactgaatt
     2161 gaagcactct aagactgaat tgaacttcaa ggaatggcaa aaggctttca ctgacgttat
     2221 gggtatggac gaattgtaca agtaaactag tagttcttct ttgagatatc gattgaacga
     2281 gctcgaattc atcgatctat attaccctgt tatccctagc gtaactcagc ttttgttccc
     2341 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4501 cgcgcacatt tccccgaaaa gtgccacctg ggtccttttc atcacgtgct ataaaaataa
     4561 ttataattta aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa
     4621 ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac
     4681 aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc
     4741 ttgtctgtgt agaagaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa
     4801 tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc
     4861 tttttgttga aattttttaa acctttgttt attttttttt cttcattccg taactcttct
     4921 accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag
     4981 taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc
     5041 gatccgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     5101 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     5161 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     5221 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     5281 tactgagagt gcagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
     5341 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
     5401 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
     5461 ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc
     5521 gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata
     5581 cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca
     5641 tgggtaagga aaagactcac gtttcgaggc cgcgattaaa ttccaacatg gatgctgatt
     5701 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     5761 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     5821 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     5881 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     5941 gcaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     6001 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     6061 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     6121 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     6181 ataagctttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     6241 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     6301 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     6361 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     6421 ttcatttgat gctcgatgag tttttctaat cagtactgac aataaaaaga ttcttgtttt
     6481 caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt aatcaaatgt
     6541 tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga agttaagtgc
     6601 gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat
     6661 t
//
