LOCUS       sequence-390775-        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for N-ter GFP11 and C-ter R9 tagged 
            mCherry..
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-mCherry
FEATURES             Location/Qualifiers
     source          1..6220
                     /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..950
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(388..406)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     656..675
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1089..1106
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1159..1177)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1173..1220
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1257..1712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1344..1363)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1554..1575
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1805..2620)
                     /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(1805..2620)
                     /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     2528..2547
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2612..2631
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2742..3330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3231..3250
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3484..3501
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3516..3655
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3581..3600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3741..3763
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3757..3948)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4757..5839)
                     /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     5801..5820
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5840..5917)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6123..6142
                     /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 atgcagtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
      301 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
      361 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      421 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
      481 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
      541 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctccagg
      601 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
      661 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
      721 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
      781 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
      841 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
      901 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggcattggta
      961 gtgggagcaa cggcagcagc ggatccgtga gccgccgtcg ccgtcgccgt cgccgtcgct
     1021 aataacattg gaagtggata acggatccga attcgagcgc cgtcgacaag cttgcggccg
     1081 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1141 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1201 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1261 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1321 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1381 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1441 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1501 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1561 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1621 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1681 gaattttaac aaactagtaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg
     1741 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1801 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1861 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1921 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     1981 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     2041 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     2101 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     2161 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2221 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatgttttca cctgaatcag
     2281 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     2341 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     2401 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     2461 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     2521 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     2581 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     2641 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     2701 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2761 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2821 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2881 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2941 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3001 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3061 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3121 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3181 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3241 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3301 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3361 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3421 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3481 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3541 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3601 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3661 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3721 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3781 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3841 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3901 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3961 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4021 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4081 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4141 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4201 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4261 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4321 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     4381 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     4441 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4501 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4561 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4621 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4681 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4741 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4801 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4861 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4921 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4981 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     5041 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5101 cgttggcaac cagcatcgca gtgggaacga taccctcatt cagcatttgc atggtttgtt
     5161 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5221 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5281 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5341 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5401 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5461 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5521 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5581 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5641 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5701 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5761 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5821 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5881 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5941 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6001 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6061 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6121 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6181 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
//
