LOCUS       sequence-361247-        4315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of Citrine-NLS in plant cell (Nucleus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4315)
  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 4315)
  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-Citrine-NLS
FEATURES             Location/Qualifiers
     source          1..4315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     354..371
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    488..509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(524..529,530..547,548..554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    562..578
                     /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     567..589
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     586..602
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     586..602
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        620..638
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     620..637
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(703..953)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    998..1022
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1027..1047)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1078..1099)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(join(1234..1746,1747..1749,1750..1752))
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     1427..1446
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1686..1707
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(1755..1779)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1823..1878
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     promoter        complement(1898..2242)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(1935..1955)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     primer_bind     complement(2318..2337)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2319..2337)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2347..2364)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2347..2363)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2356..2378)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2572..2591)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2691..2713
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2751..2769)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2943..2962)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2954..3769
                     /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     complement(3027..3046)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3927..4315,1..200)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
       61 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      241 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      301 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      361 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      541 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      601 accatgatta cgccatgcta tttaggtgac actatagaat acgcggccgc aagcttgcat
      661 gcctgcaggt cgactctaga gcggatccac aattgcggcc gcatctagta acatagatga
      721 caccgcgcgc gataatttat cctagtttgc gcgctatatt ttgttttcta tcgcgtatta
      781 aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta
      841 catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag
      901 accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac gattctagat
      961 actagtgcta gcccatgggg agcctcctga gcctccaacc actttgtaca agaaagctgg
     1021 gtctcacacc ttgcgcttct tcttaggagg cttgtacaaa gtggtcccgg cggcggtcac
     1081 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
     1141 gtagctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     1201 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     1261 caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt
     1321 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     1381 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     1441 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     1501 gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag cacatcaggc cgtagccgaa
     1561 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     1621 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     1681 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     1741 cttgctcacc atggagcctg cttttttgta caaacttgtg gagcctcctg agcctccaag
     1801 cttggtaccg agctcgaatt ctgtaattgt aaatagtaat tgtaatgttg tttgttgttt
     1861 gttgttgttg gtaattgttg aaggatctca agaagcctgt cctctccaaa tgaaatgaac
     1921 ttccttatat agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt
     1981 cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt
     2041 ccacgatgtt cctcgtgggt gggggtccat ctttgggacc actgtcggta gaggcatctt
     2101 gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggagccacct tccttttcta
     2161 ctgtcctttc gataagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat
     2221 taccctttgt tgaaaagtct caatagccct cggtcttctg agcccgggag atctggcctc
     2281 gagatcccgg gtaccgagct cgaattctgc ggccgcgccc tatagtgagt cgtattacac
     2341 tagctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2401 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2461 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctgt agtaatattt
     2521 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     2581 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     2641 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2701 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     2761 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     2821 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2881 gtatccgctc atgagacaat aaccctgata aatgcttatg tctgcttaca taaacagtaa
     2941 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcaaggc cgcgattaaa
     3001 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     3061 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     3121 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     3181 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     3241 actcacctct gcgatcccag ggaaaacagc attccaggta ttagaagaat atcctgattc
     3301 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     3361 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     3421 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     3481 acaagtctgg aaagaaatgc ataagctatt gccattctca ccggattcag tcgtcactca
     3541 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     3601 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     3661 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     3721 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat taaaacatat
     3781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtg
//
