LOCUS       sequence-483311-        6297 bp    DNA     circular SYN 15-AUG-2025
DEFINITION  For stably expressing fusion proteins with N terminal TurboID for 
            proximity labeling in plants .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6297)
  AUTHORS   Greenhalgh R, Horner W, Klimpel KA, Chatterjee S, Scarpin MR, 
            Brunkard JO
  TITLE     Seamless, modular binary vectors for plant cell and molecular 
            biology.
  JOURNAL   Plant Biotechnol J. 2025 Jun 26. doi: 10.1111/pbi.70233.
  PUBMED    40574375
REFERENCE   2  (bases 1 to 6297)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Biotechnol J. 2025 Jun 26. doi: 10.1111/pbi.70233."
COMMENT     Sequence Label: pAVEC1_N-TurboID
FEATURES             Location/Qualifiers
     source          1..6297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     368..387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     621..638
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    653..792
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(718..737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     878..900
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(894..1085)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1894..2976)
                     /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     2938..2957
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2977..3054)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3260..3279
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3367..3386
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3367..3385
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3386..3410
                     /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             3425..3447
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3491..3508
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3515..3808
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4139..4165
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4524..4541
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4594..4612)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4608..4655
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4692..5147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4779..4798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4989..5010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5239..6054)
                     /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(5239..6054)
                     /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     5962..5981
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6046..6065
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6176..6297,1..467)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
       61 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      121 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      181 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
      241 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
      301 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      361 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      421 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      481 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      541 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      601 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
      661 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
      721 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
      781 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      841 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      901 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
      961 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     1021 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     1081 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     1141 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     1201 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     1261 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     1321 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     1381 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     1441 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     1501 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     1561 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     1621 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     1681 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     1741 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     1801 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     1861 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1921 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1981 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     2041 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     2101 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     2161 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     2221 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     2281 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     2341 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     2401 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     2461 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     2521 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     2581 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     2641 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     2701 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     2761 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     2821 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     2881 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     2941 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     3001 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     3061 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     3121 cgttgagcac cgccgccaca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     3181 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     3241 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     3301 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     3361 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     3421 taattttgtt taactttaag aaggagatat acatatgggc agcagccatc atcatcatca
     3481 cggcagcggc ctggtgccgc gcggcagcgc tagcatgtcg gactcagaag tcaatcaaga
     3541 agctaagcca gaggtcaagc cagaagtcaa gcctgagact cacatcaatt taaaggtgtc
     3601 cgatggatct tcagagatct tcttcaagat caaaaagacc actcctttaa gaaggctgat
     3661 ggaagcgttc gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgg
     3721 tattagaatt caagctgatc agacccctga agatttggac atggaggata acgatattat
     3781 tgaggctcac agagaacaga ttggtggtat gttcaaattg aacacgaaaa acatcaaggt
     3841 tttaacgccg tctggattca aatctttctc tggtatccag aaagtctata aaccgttcta
     3901 ccaccatatc atttttgacg acgggtcgga gatcaagtgt tctgacaatc attcctttgg
     3961 aaaggataaa atcaaagcgt cgacgatcaa agtgggcgac tatttacagg gaaaaaaagt
     4021 actttataat gagatcgtag aggaaggaat ttatctgtac gacctgctta acgtggggga
     4081 agataactta tattacacta atggaatcgt cagccataac tgcgaagcct cggggaagta
     4141 cccttacgac gttcctgact acgctggcgc cgagtattgc ctgtcttacg acacagagat
     4201 tctgaccgtt gaatatggat tccttcctat cggtaagatc gtggaggaac ggattgaatg
     4261 cacagtctat acggtagata aaaatggctt tgtgtataca caacctattg ctcagtggca
     4321 taaccgggga gaacaggaag ttttcgaata ctgcttagaa gacggttcga ttatccgtgc
     4381 aacgaaagat cacaaatttc tgacgaccga cggtcagctg ttaccgattg atgagatttt
     4441 cgaacggggg ttagacctga aacaagttga tggtttgccg taagaattcg agctccgtcg
     4501 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     4561 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     4621 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     4681 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4741 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4801 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4861 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4921 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4981 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5041 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5101 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5161 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5221 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     5281 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     5341 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     5401 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     5461 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     5521 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     5581 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     5641 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     5701 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     5761 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     5821 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     5881 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     5941 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     6001 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     6061 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     6121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttca
//
