LOCUS       sequence-213488-        5894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid expresses mCherry fused to Lifeact peptide. It labels 
            actin bundles and filopodia in Capsaspora. Codons have been 
            optimised according to their usages in Capsaspora and C. 
            fragrantissima..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5894)
  AUTHORS   Parra-Acero H, Ros-Rocher N, Perez-Posada A, Kozyczkowska A, 
            Sanchez-Pons N, Nakata A, Suga H, Najle SR, Ruiz-Trillo I
  TITLE     Transfection of Capsaspora owczarzaki, a close unicellular relative 
            of animals.
  JOURNAL   Development. 2018 May 11. pii: dev.162107. doi: 10.1242/dev.162107.
  PUBMED    29752387
REFERENCE   2  (bases 1 to 5894)
  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: 
            "Development. 2018 May 11. pii: dev.162107. doi: 
            10.1242/dev.162107."
COMMENT     Sequence Label: pONSY-Lifeact:mCherry
FEATURES             Location/Qualifiers
     source          1..5894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    95..116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(131..136,137..154,155..161)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    169..185
                     /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)."
     primer_bind     174..196
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     193..209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     193..209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1153..1203
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             1228..1938
                     /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(1373..1391)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1641..1660
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1835..1858
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2315..2334)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2316..2334)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2341..2358)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2341..2357)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2350..2372)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2498..2926
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2585..2604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2795..2816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3270..4064
                     /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(3324..3343)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3934..3953
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4082..4150,4151..4942)
                     /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     complement(4300..4319)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5113..5701
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5602..5621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5855..5872
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
       61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      121 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      181 aacaatttca cacaggaaac agctatgacc atgattacgc caagcttggt accaaatgca
      241 cagttagcaa cgaccgacaa tcagcaacct ccaaactctc agtttttttc ccccaatcgt
      301 tgtcgattta gcttttgatg ctgcattttg aaatagaacg acaaccgaat ttgggagaga
      361 gcactctttt acaggtttgt gaaaagaaaa aaaatcgaac gtgacttgct tccatcttcc
      421 ttctcgaagc cgatctcggc ccatccccgc ttgtcccctc ctcccctcct cgcaatccgc
      481 tagacagtgt ctctgcacac tggattgcgg tgcgcgggcc atccgatgcc cctttcctcg
      541 gcctggagat ggacgtgaaa gtggaaaacg aaaaacgcga aacagaaaca tgtggttctt
      601 ttttttttct ttttcttttt cgccttcggg cttttctctc tcgcttcctc ttctccgctc
      661 tcttcttcgc gctcttttgc ctcttgcgtg agatgctcgt cttgcaatcg tctgccactg
      721 ctgcgctgtc acgatcgatc gcatcggaca agccgtttct gttttgaaat ttagaacgtt
      781 ctggaaaaca acaaaaaatt gccttttttt cacttttttc ttttttcgtt tttttccctc
      841 ttcaacactt cctccaactg tcgttctgcg tttgttcttc gctctcgtcg tgcacactcg
      901 gattaggatc cattcatccc catccccttg cgcattgcca aaaacgagcg tcgaaacgcg
      961 aacgtcagcg tggggtgttg acgccgaacc ccacgagtgt ttttttttaa aatgctgttt
     1021 catcgttttc ttccttctcc ccacgtgcaa tcctgattta aataattgtc gctttttgat
     1081 ttggaaatgc tcactcttcc atttttttat tatttaacac acacagaaca accttcacag
     1141 gatcccggga ccatgggtgt ggcagacctg attaagaagt tcgagagcat tagcaaggag
     1201 gagggtgacc caccatctag tgatatcatg gtgagcaagg gcgaggagga caacatggcc
     1261 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     1321 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1381 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1441 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1501 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1561 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1621 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1681 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1741 ctgaaggacg gcggccacta cgacgccgag gtcaagacca cctacaaggc caagaagccc
     1801 gtgcagctgc ccggcgccta caacgtcaac atcaagctgg acatcacctc ccacaacgag
     1861 gactacacca tcgtggaaca gtacgagcgc gccgagggcc gccactccac cggcggcatg
     1921 gacgagctgt acaagtaaat tttgtgtttg ccaagacaca tttgtttatt tcaatcattt
     1981 ctaagaccaa aaagggattt catgattttt gttgcttgca ttgaagtttt ttttggttgt
     2041 catttttttt gcgttggtta tgagcgcaat atattccatt atggcaagaa acacgacgag
     2101 ttgtttttac agtggaggga tgagcaagga atggggaaat ggagaacaag tggaatgaaa
     2161 tggtttgact ttgtgcggcg aaagtgttgc cattttcaat catgctgcgc ctggcaccta
     2221 gatgcgtgtg attgctcatt gctagtgctg ttctcaccgc ggtcatccat cacactggcg
     2281 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt attacaattc
     2341 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     2401 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     2461 cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta
     2521 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2581 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2641 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2701 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2761 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2821 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2881 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaattcag
     2941 ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag aaacggtgct
     3001 gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga
     3061 gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga
     3121 cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca
     3181 aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg
     3241 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3301 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3361 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3421 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3481 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3541 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atcccacctt gctcctgccg
     3601 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3661 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3721 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3781 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3841 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3901 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3961 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4021 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattgaa aaaggaagag
     4081 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4141 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4201 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4261 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4321 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4381 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4441 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4501 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4561 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4621 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4681 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4741 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4801 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4861 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4921 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4981 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5041 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5101 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5161 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5221 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5281 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5341 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5401 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5461 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5521 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5581 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5641 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5701 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5761 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5821 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5881 agagcgccca atac
//
