LOCUS       sequence-390763-        5566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for N-ter GFP11 and C-ter R9 tagged 
            lifeact peptide..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5566)
  AUTHORS   Wang JW, Squire HJ, Goh NS, Ni HM, Lien E, Wong C, Gonzalez-Grandio 
            E, Landry MP
  TITLE     Delivered complementation in planta (DCIP) enables measurement of 
            peptide-mediated protein delivery efficiency in plants.
  JOURNAL   Commun Biol. 2023 Aug 12;6(1):840. doi: 10.1038/s42003-023-05191-5.
  PUBMED    37573467
REFERENCE   2  (bases 1 to 5566)
  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: "Commun 
            Biol."; date: "2023-08-12"; volume: "6(1):840. doi"; pages: " 
            10.1038/s42003-023-05191-5"
COMMENT     Sequence Label: 1BR9-Lifeact
FEATURES             Location/Qualifiers
     source          1..5566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..25
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6..24
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    25..49
                     /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)."
     RBS             64..86
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             105..122
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             123..143
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             150..197
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             246..296
                     /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             435..452
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(505..523)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      519..566
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      603..1058
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(690..709)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     900..921
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1151..1966)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1151..1966)
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1874..1893
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1958..1977
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2088..2676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2577..2596
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2830..2847
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2862..3001
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2927..2946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3087..3109
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3103..3294)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4103..5185)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5147..5166
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5186..5263)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5469..5488
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
       61 aattttgttt aactttaaga aggagatata ccatgggttc ttctcaccat caccatcacc
      121 atgaaaacct gtacttccaa tccaatgcac gcgatcacat ggtcctgcac gagtacgtga
      181 acgccgccgg gatcactggt ggcggaggtt ctggaggcgg tggatcgtac ttccaatcca
      241 atgcaatggg cgtggccgac ctgatcaaga agttcgagag catcagcaag gaagagggca
      301 ttggtagtgg gagcaacggc aacagcggat ccgtgagccg ccgtcgccgt cgccgtcgcc
      361 gtcgctaata acattggaag tggataacgg atccgaattc gagcgccgtc gacaagcttg
      421 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
      481 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
      541 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
      601 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
      661 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      721 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      781 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      841 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
      901 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
      961 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1021 taacgcgaat tttaacaaac tagtaacgtt tacaatttca ggtggcactt ttcggggaaa
     1081 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1141 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1201 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1261 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1321 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1381 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1441 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1501 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     1561 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaatg ttttcacctg
     1621 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1681 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     1741 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     1801 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     1861 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     1921 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     1981 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2041 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2101 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2161 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2221 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2281 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2341 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2401 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2461 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2521 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2581 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2641 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2701 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2761 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2821 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     2881 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     2941 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3001 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3061 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3121 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3181 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3241 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3301 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3361 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3421 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     3481 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     3541 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3601 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     3661 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     3721 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     3781 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     3841 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     3901 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     3961 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4021 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4081 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4141 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4201 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4261 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4321 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4381 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4441 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatacc ctcattcagc atttgcatgg
     4501 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4561 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4621 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     4681 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     4741 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     4801 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     4861 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     4921 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     4981 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5041 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5101 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5161 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5221 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5281 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5341 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5401 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5461 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     5521 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgc
//
