LOCUS       sequence-405826-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for N-ter GFP11 and C-ter R9 tagged 
            tagBFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  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 6220)
  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-BFP
FEATURES             Location/Qualifiers
     source          1..6220
                     /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..1245
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             1384..1401
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1454..1472)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1468..1515
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1552..2007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1639..1658)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1849..1870
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2100..2915)
                     /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(2100..2915)
                     /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     2823..2842
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2907..2926
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3037..3625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3526..3545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3779..3796
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3811..3950
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3876..3895)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4036..4058
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4052..4243)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5052..6134)
                     /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     6096..6115
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6135..6212)
                     /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 ttaatggcat tggtagtggg
     1261 agcaacggca gcagcggatc cgtgagccgc cgtcgccgtc gccgtcgccg tcgctaataa
     1321 cattggaagt ggataacgga tccgaattcg agcgccgtcg acaagcttgc ggccgcactc
     1381 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1441 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1501 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1561 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1621 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1681 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1741 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1801 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1861 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1921 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1981 ttaacaaact agtaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa
     2041 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     2101 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2161 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2221 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2281 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2341 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     2401 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2461 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2521 tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt tttcacctga atcaggatat
     2581 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2641 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2701 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2761 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2821 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2881 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2941 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3001 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3061 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3121 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3181 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3241 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3301 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3361 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3421 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3481 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3541 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3601 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3661 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3721 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3781 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3841 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3901 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3961 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4021 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4081 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4141 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4201 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4261 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4321 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4381 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4441 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4501 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4561 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4621 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4681 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4741 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4801 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4861 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4921 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4981 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5041 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5101 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5161 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5221 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5281 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5341 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5401 gcaaccagca tcgcagtggg aacgataccc tcattcagca tttgcatggt ttgttgaaaa
     5461 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5521 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5581 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5641 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5701 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5761 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5821 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5881 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5941 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6001 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6061 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6121 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6181 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
//
