LOCUS       sequence-390949-        6004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for Hisx6 tagged GFP1-10..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6004)
  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 6004)
  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: 1B-GFP1-10
FEATURES             Location/Qualifiers
     source          1..6004
                     /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"
     primer_bind     complement(189..217)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             873..890
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(943..961)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      957..1004
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1041..1496
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1128..1147)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1338..1359
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1589..2404)
                     /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(1589..2404)
                     /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     2312..2331
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2396..2415
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2526..3114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3015..3034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3268..3285
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3300..3439
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3365..3384)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3525..3547
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3541..3732)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4541..5623)
                     /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     5585..5604
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5624..5701)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5907..5926
                     /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 tccaatgcca ccatgatcga tagcaaagga gaagaacttt
      181 tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt
      241 ctgtccgtgg agagggtgaa ggtgatgcta caattggaaa actcaccctt aaatttattt
      301 gcactactgg aaaactacct gttccatggc caacacttgt cactactctg acctatggtg
      361 ttcaatgctt ttcccgttat ccggatcaca tgaaacggca tgactttttc aagagtgcca
      421 tgcccgaagg ttatgtacag gaacgcacta tatctttcaa agatgacggg aaatacaaga
      481 cgcgtgctgt cgtcaagttt gaaggtgata cccttgttaa tcgtatcgag ttaaaaggta
      541 ctgattttaa agaagatgga aacattctcg gacacaaact cgagtacaac tttaactcac
      601 acaatgtata catcacggca gacaaacaaa agaatggaat caaagctaac ttcaccgttc
      661 gccacaacgt tgaagatggt tccgttcaac tagcagacca ttatcaacaa aatactccaa
      721 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtcgacacaa actgtccttt
      781 cgaaagatcc caacgaaaag tgataataac attggaagtg gataacggat ccgaattcga
      841 gcgccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
      901 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
      961 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1021 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1081 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1141 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1201 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1261 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1321 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1381 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1441 gctgatttaa caaaaattta acgcgaattt taacaaacta gtaacgttta caatttcagg
     1501 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1561 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     1621 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1681 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1741 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     1801 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     1861 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     1921 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     1981 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2041 caacaatgtt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2101 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2161 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2221 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2281 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2341 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2401 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     2461 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2521 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2581 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2641 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2701 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2761 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2821 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2881 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2941 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3001 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3061 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3121 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3181 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3241 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3301 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3361 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3421 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3481 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3541 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3601 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3661 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     3721 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     3781 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     3841 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     3901 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     3961 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4021 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4081 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4141 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4201 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4261 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4321 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4381 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4441 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4501 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4561 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4621 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     4681 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     4741 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     4801 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4861 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgataccct
     4921 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     4981 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5041 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5101 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5161 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5221 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5281 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5341 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5401 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5461 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5521 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     5581 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     5641 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     5701 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     5761 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5821 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     5881 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     5941 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6001 ccgc
//
