LOCUS       sequence-405823-        6223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for N-ter GFP11 R9 tagged BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6223)
  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 6223)
  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-BFPSTOP
FEATURES             Location/Qualifiers
     source          1..6223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     198..217
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     301..320
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        301..319
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    320..344
                     /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             359..381
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             400..417
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             418..438
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             445..492
                     /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             550..1248
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             1387..1404
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1457..1475)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1471..1518
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1555..2010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1642..1661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1852..1873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2103..2918)
                     /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(2103..2918)
                     /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     2826..2845
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2910..2929
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3040..3628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3529..3548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3782..3799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3814..3953
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3879..3898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4039..4061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4055..4246)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5055..6137)
                     /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     6099..6118
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6138..6215)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
       61 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
      121 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
      181 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
      241 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
      301 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      361 tgtttaactt taagaaggag atataccatg ggttcttctc accatcacca tcaccatgaa
      421 aacctgtact tccaatccaa tgcacgcgat cacatggtcc tgcacgagta cgtgaacgcc
      481 gccgggatca ctggtggcgg aggttctgga ggcggtggat cgtacttcca atccaatgca
      541 gcttcctcca gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc
      601 gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
      661 cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg
      721 gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac
      781 ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac
      841 gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac
      901 gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc
      961 ggctgggagg ccttcaccga aacgctgtac cccgctgacg gcggcctgga aggcagaaac
     1021 gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat
     1081 agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga
     1141 ctggaaagaa tcaaggaggc caacaacgaa acctacgtcg agcagcacga ggtggcagtg
     1201 gccagatact gcgacctccc tagcaaactg gggcacaagc ttaattgagg cattggtagt
     1261 gggagcaacg gcaacagcgg atccgtgagc cgccgtcgcc gtcgccgtcg ccgtcgctaa
     1321 taacattgga agtggataac ggatccgaat tcgagcgccg tcgacaagct tgcggccgca
     1381 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1441 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1501 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1561 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1621 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1681 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1741 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1801 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1861 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1921 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1981 attttaacaa actagtaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     2041 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2101 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2161 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2221 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2281 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2341 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2401 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2461 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2521 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tgttttcacc tgaatcagga
     2581 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2641 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2701 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2761 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2821 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2881 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     2941 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3001 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3061 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3181 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3421 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3661 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3721 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3781 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3841 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3901 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3961 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4021 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4081 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4141 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4201 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4261 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4321 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4381 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4441 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4501 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4561 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4621 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4681 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4741 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4801 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4861 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4921 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4981 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5041 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5101 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5161 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5221 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5281 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     5341 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5401 ttggcaacca gcatcgcagt gggaacgata ccctcattca gcatttgcat ggtttgttga
     5461 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5521 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5581 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5641 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5701 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5761 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5821 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5881 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5941 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6001 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6061 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6121 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6181 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tct
//
