LOCUS       sequence-213708-        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    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1165..1215
                     /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             1240..1950
                     /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(1385..1403)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1653..1672
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1847..1870
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2327..2346)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2328..2346)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2353..2370)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2353..2369)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2362..2384)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2510..2938
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2597..2616)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2807..2828
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3282..4076
                     /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(3336..3355)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3946..3965
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4094..4162,4163..4954)
                     /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(4312..4331)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5125..5713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5614..5633
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5867..5884
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccaaatg cacagttagc aacgaccgac aatcagcaac ctccaaactc tcagtttttt
      301 tcccccaatc gttgtcgatt tagcttttga tgctgcattt tgaaatagaa cgacaaccga
      361 atttgggaga gagcactctt ttacaggttt gtgaaaagaa aaaaaatcga acgtgacttg
      421 cttccatctt ccttctcgaa gccgatctcg gcccatcccc gcttgtcccc tcctcccctc
      481 ctcgcaatcc gctagacagt gtctctgcac actggattgc ggtgcgcggg ccatccgatg
      541 cccctttcct cggcctggag atggacgtga aagtggaaaa cgaaaaacgc gaaacagaaa
      601 catgtggttc tttttttttt ctttttcttt ttcgccttcg ggcttttctc tctcgcttcc
      661 tcttctccgc tctcttcttc gcgctctttt gcctcttgcg tgagatgctc gtcttgcaat
      721 cgtctgccac tgctgcgctg tcacgatcga tcgcatcgga caagccgttt ctgttttgaa
      781 atttagaacg ttctggaaaa caacaaaaaa ttgccttttt ttcacttttt tcttttttcg
      841 tttttttccc tcttcaacac ttcctccaac tgtcgttctg cgtttgttct tcgctctcgt
      901 cgtgcacact cggattagga tccattcatc cccatcccct tgcgcattgc caaaaacgag
      961 cgtcgaaacg cgaacgtcag cgtggggtgt tgacgccgaa ccccacgagt gttttttttt
     1021 aaaatgctgt ttcatcgttt tcttccttct ccccacgtgc aatcctgatt taaataattg
     1081 tcgctttttg atttggaaat gctcactctt ccattttttt attatttaac acacacagaa
     1141 caaccttcac aggatcccgg gaccatgggt gtggcagacc tgattaagaa gttcgagagc
     1201 attagcaagg aggagggtga cccaccatct agtgatatca tggtgagcaa gggcgaggag
     1261 gacaacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     1321 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1381 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1441 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1501 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1561 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1621 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1681 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1741 cagaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacaag
     1801 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagct ggacatcacc
     1861 tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc
     1921 accggcggca tggacgagct gtacaagtaa attttgtgtt tgccaagaca catttgttta
     1981 tttcaatcat ttctaagacc aaaaagggat ttcatgattt ttgttgcttg cattgaagtt
     2041 ttttttggtt gtcatttttt ttgcgttggt tatgagcgca atatattcca ttatggcaag
     2101 aaacacgacg agttgttttt acagtggagg gatgagcaag gaatggggaa atggagaaca
     2161 agtggaatga aatggtttga ctttgtgcgg cgaaagtgtt gccattttca atcatgctgc
     2221 gcctggcacc tagatgcgtg tgattgctca ttgctagtgc tgttctcacc gcggtcatcc
     2281 atcacactgg cggccgctcg agcatgcatc tagagggccc aattcgccct atagtgagtc
     2341 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2401 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2461 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt
     2521 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2581 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2641 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2701 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2761 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2821 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2881 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2941 aacaaaattc agggcgcaag ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc
     3001 agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg
     3061 caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg
     3121 cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg
     3181 ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg
     3241 gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3301 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3361 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3421 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3481 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3541 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatcccacc
     3601 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3661 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3721 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3781 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3841 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3901 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3961 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4021 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaattg
     4081 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4141 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4201 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4261 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4321 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     4381 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4441 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4501 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4561 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4621 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4681 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4741 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4801 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4861 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4921 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4981 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5041 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5101 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5161 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5221 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5281 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5341 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5401 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5461 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5521 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5581 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5641 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5701 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5761 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5821 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5881 cgaggaagcg gaag
//
