LOCUS       sequence-390853-        5509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for N-ter GFP11 and C-ter R9 tagged 
            proteins. Empty backbone for LIC cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5509)
  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 5509)
  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
FEATURES             Location/Qualifiers
     source          1..5509
                     /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             378..395
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(448..466)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      462..509
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      546..1001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(633..652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     843..864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1094..1909)
                     /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(1094..1909)
                     /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     1817..1836
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1901..1920
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2031..2619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2520..2539
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2773..2790
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2805..2944
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2870..2889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3030..3052
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3046..3237)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4046..5128)
                     /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     5090..5109
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5129..5206)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5412..5431
                     /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 atattggtag tgggagcaac ggcaacagcg gatccgtgag ccgccgtcgc cgtcgccgtc
      301 gccgtcgcta ataacattgg aagtggataa cggatccgaa ttcgagcgcc gtcgacaagc
      361 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
      421 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      481 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
      541 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
      601 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      661 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      721 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      781 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      841 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      901 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
      961 atttaacgcg aattttaaca aactagtaac gtttacaatt tcaggtggca cttttcgggg
     1021 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1081 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     1141 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     1201 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     1261 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     1321 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     1381 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     1441 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     1501 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atgttttcac
     1561 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     1621 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     1681 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     1741 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     1801 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     1861 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     1921 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     1981 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2041 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2101 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2161 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2221 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2281 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2341 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2401 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2461 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2521 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2581 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2641 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2701 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2761 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     2821 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     2881 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     2941 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3001 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3061 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3121 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3181 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3241 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3301 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     3361 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     3421 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     3481 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     3541 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     3601 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     3661 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     3721 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     3781 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     3841 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     3901 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     3961 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4021 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4081 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4141 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4201 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4261 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4321 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4381 ccatctgatc gttggcaacc agcatcgcag tgggaacgat accctcattc agcatttgca
     4441 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4501 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4561 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     4621 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     4681 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     4741 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     4801 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     4861 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     4921 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     4981 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5041 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5101 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5161 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5221 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5281 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5341 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5401 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     5461 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgc
//
