LOCUS       sequence-361182-        4537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of RBCS1a(1–79)-TagRFP in plant cell 
            (Plastid/Chloroplast).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4537)
  AUTHORS   Osaki Y, Kodama Y
  TITLE     Particle bombardment and subcellular protein localization analysis 
            in the aquatic plant Egeria densa.
  JOURNAL   PeerJ. 2017 Sep 7;5:e3779. doi: 10.7717/peerj.3779. eCollection 
            2017.
  PUBMED    28894649
REFERENCE   2  (bases 1 to 4537)
  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: "PeerJ."; 
            date: "2017-09-7"; volume: "5:e3779. doi"; pages: " 
            10.7717/peerj.3779. eCollection 2017"
COMMENT     Sequence Label: pGWT35S-RBCS1a(1–79)-TagRFP
FEATURES             Location/Qualifiers
     source          1..4537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..47)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(201..789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(281..300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(947..1762)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTSAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1670..1689
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1754..1773
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1947..1965
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2003..2025)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2125..2144
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2338..2360
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2352..2369
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2353..2369
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2379..2398
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2379..2397
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2474..2818
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     2761..2781
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     misc_feature    2838..2893
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     protein_bind    2937..2961
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3201..3914
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     protein_bind    complement(3916..3940)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3985..4235
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        complement(4300..4318)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4301..4318)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4336..4352)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4336..4352)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4349..4371)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4360..4376
                     /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(4384..4390,4391..4408,4409..4414))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4429..4450
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
       61 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      361 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      601 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
      661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
      781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
      841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
      901 aattaaaaat gaagttttaa atcaatctaa agtatatatg ttttaattag aaaaactcat
      961 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1021 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1081 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1141 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1201 atggcaatag cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1261 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1321 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1381 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1441 ggaatgctgt tttccctggg atcgcagagg tgagtaacca tgcatcatca ggagtacgga
     1501 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     1561 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     1621 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     1681 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctt gagcaagacg
     1741 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacataa
     1801 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     1861 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     1921 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     1981 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     2041 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     2101 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     2161 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ttactacagg cgccattcgc
     2221 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     2281 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2341 agtcacgacg ttgtaaaacg acggccagtg agctagtgta atacgactca ctatagggcg
     2401 cggccgcaga attcgagctc ggtacccggg atctcgaggc cagatctccc gggctcagaa
     2461 gaccgagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc
     2521 attgcccagc tatctgtcac ttatcgaaag gacagtagaa aaggaaggtg gctcctacaa
     2581 atgccatcat tgcgataaag gaaaggctat cgttcaagat gcctctaccg acagtggtcc
     2641 caaagatgga cccccaccca cgaggaacat cgtggaaaaa gaagacgttc caaccacgtc
     2701 ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca
     2761 ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agaggacagg
     2821 cttcttgaga tccttcaaca attaccaaca acaacaaaca acaaacaaca ttacaattac
     2881 tatttacaat tacagaattc gagctcggta ccaagcttgg aggctcagga ggctccacaa
     2941 gtttgtacaa aaaagcaggc ttcatggctt cctctatgct ctcttccgct actatggttg
     3001 cctctccggc tcaggccact atggtcgctc ctttcaacgg acttaagtcc tccgctgcct
     3061 tcccagccac ccgcaaggct aacaacgaca ttacttccat cacaagcaac ggcggaagag
     3121 ttaactgcat gcaggtgtgg cctccgattg gaaagaagaa gtttgagact ctctcttacc
     3181 ttcctgacct taccgattcc atggtgtcta agggcgaaga gctgattaag gagaacatgc
     3241 acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg
     3301 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     3361 ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc agaaccttca
     3421 tcaaccacac ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat
     3481 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     3541 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg
     3601 gccctgtgat gcagaagaaa acactcggct gggaggccaa caccgagatg ctgtaccccg
     3661 ctgacggcgg cctggaaggc agaaccgaca tggccctgaa gctcgtgggc gggggccacc
     3721 tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc
     3781 ccggcgtcta ctatgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct
     3841 acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc
     3901 acaaacttaa ttgagaccca gctttcttgt acaaagtggt tggaggctca ggaggctccc
     3961 catgggctag cactagtatc tagaatcgtt caaacatttg gcaataaagt ttcttaagat
     4021 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
     4081 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
     4141 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
     4201 aattatcgcg cgcggtgtca tctatgttac tagatgcggc cgcaattgtg gatccgctct
     4261 agagtcgacc tgcaggcatg caagcttgcg gccgcgtatt ctatagtgtc acctaaatag
     4321 catggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4381 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4441 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4501 gctgcattaa tgaatcggcc aacgcgcggg gagaggc
//
