LOCUS       sequence-370396-        4013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To chromosomally tag genes of interest with mCherry. 
            FRT-mCherry-kan-FRT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4013)
  AUTHORS   Avraham, Roi
  TITLE     Bacterial fluorescent reporters and tags toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4013)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKD4_mCherry
FEATURES             Location/Qualifiers
     source          1..4013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(70..117)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             477..1271
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(531..550)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1141..1160
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(1462..1495)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(1533..2240)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDREFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1811..1830)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2080..2098
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      2255..2643
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(2745..3536,3537..3605))
                     /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     3368..3387
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3606..3710)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3800..3886
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3978..4005
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cagtgccaag cttgcatgca gattgcagca ttacacgtct tgagcgattg tgtaggctgg
       61 agctgcttcg aagttcctat actttctaga gaataggaac ttcggaatag gaacttcaag
      121 atcccccacg ctgccgcaag cactcagggc gcaagggctg ctaaaggaag cggaacacgt
      181 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
      241 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
      301 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
      361 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
      421 tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga
      481 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
      541 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
      601 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
      661 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
      721 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
      781 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
      841 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
      901 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
      961 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     1021 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     1081 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     1141 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     1201 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     1261 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     1321 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     1381 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     1441 ccccagcttc aaaagcgctc tgaagttcct atactttcta gagaatagga acttcggaat
     1501 aggaactaag gaggatattc atatggacca tgttacttgt acagctcgtc catgccgccg
     1561 gtggagtggc ggccctcggc gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg
     1621 gaggtgatgt ccaacttgat gttgacgttg taggcgccgg gcagctgcac gggcttcttg
     1681 gccttgtagg tggtcttgac ctcagcgtcg tagtggccgc cgtccttcag cttcagcctc
     1741 tgcttgatct cgcccttcag ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc
     1801 cagcccatgg tcttcttctg cattacgggg ccgtcggagg ggaagttggt gccgcgcagc
     1861 ttcaccttgt agatgaactc gcggtcctgc agggaggagt cctgggtcac ggtcaccacg
     1921 ccgccgtcct cgaagttcat cacgcgctcc cacttgaagc cctcggggaa ggacagcttc
     1981 aagtagtcgg ggatgtcggc ggggtgcttc acgtaggcct tggagccgta catgaactga
     2041 ggggacagga tgtcccaggc gaagggcagg gggccaccct tggtcacctt cagcttggcg
     2101 gtctgggtgc cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg
     2161 ttcacggagc cctccatgtg caccttgaag cgcatgaact ccttgatgat ggccatgtta
     2221 tcctcctcgc ccttgctcac caggctaatt cccatgtcag ccgttaagtg ttcctgtgtc
     2281 actcaaaatt gctttgagag gctctaaggg cttctcagtg cgttacatcc ctggcttgtt
     2341 gtccacaacc gttaaacctt aaaagcttta aaagccttat atattctttt ttttcttata
     2401 aaacttaaaa ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac
     2461 atgagagctt agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg
     2521 ttagccatga gggtttagtt cgttaaacat gagagcttag tacgttaaac atgagagctt
     2581 agtacgtgaa acatgagagc ttagtacgta ctatcaacag gttgaactgc tgatcttcag
     2641 atcctctacg ccggacgcat cgtggccgga tcttgcggcc gcaaaaatta aaaatgaagt
     2701 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2761 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2821 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2881 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2941 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3001 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3061 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3121 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3181 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3241 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3301 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3361 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3421 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3481 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3541 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3601 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3661 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3721 cgaaaagtgc cacctgcatc gatggccccc gatggtagtg tggggtctcc ccatgcgaga
     3781 gtagggaact gccaggcatc aaataaaatg aaaggctcag tcgaaagact gggcctttcg
     3841 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     3901 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     3961 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ggc
//
