LOCUS       sequence-229865-        6834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6834)
  AUTHORS   Pavicic M, Mouhu K, Wang F, Bilicka M, Chovancek E, Himanen K
  TITLE     Genomic and Phenomic Screens for Flower Related RING Type Ubiquitin 
            E3 Ligases in Arabidopsis.
  JOURNAL   Front Plant Sci. 2017 Mar 28;8:416. doi: 10.3389/fpls.2017.00416. 
            eCollection 2017.
  PUBMED    28400782
REFERENCE   2  (bases 1 to 6834)
  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: "Front Plant
            Sci."; date: "2017-03-28"; volume: "8:416. doi"; pages: " 
            10.3389/fpls.2017.00416. eCollection 2017"
COMMENT     Sequence Label: pDEST 565-XERICO
FEATURES             Location/Qualifiers
     source          1..6834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             123..770
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="PILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFE
                     LGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV
                     SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYM
                     DPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     728..750
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             777..794
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    801..825
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1318..1342)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1402..1434
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1441..1458
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1624..1710
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1802..1829
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1883..1901)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1897..1944
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1981..2436
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2068..2087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2278..2299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2551..3342,3343..3411))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3174..3193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3412..3504)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3585..4173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4074..4093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4327..4344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4359..4498
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4424..4443)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4584..4606
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4600..4791)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5363..6445)
                     /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     6407..6426
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6446..6523)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6729..6748
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatccca tcaccatcac catcacggcg
      121 cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg
      181 aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc
      241 gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg
      301 atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt
      361 tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata
      421 ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt
      481 ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat
      541 atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg
      601 ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac
      661 gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc
      721 ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg
      781 ttccgcgtgg atctgatatc acaagtttgt acaaaaaagc aggctctatg ggtctatcaa
      841 gtcttcctgg tccatcagaa ggaatgttat gtgtgatatt agttaataca gcattatcga
      901 tctccattgt caaaggcatt gtaagatcat tccttggcat agtaggaatc agtctctcgc
      961 cgtcttcatc ctcgccttct tcggtgacgg tatcttcaga gaattcatca acttcagagt
     1021 catttgattt ccgggtctgc caaccagaga gttaccttga ggagttcagg aaccggactc
     1081 cgacactgag gtttgagagc ttgtgcaggt gcaagaaaca ggcagacaat gagtgttctg
     1141 tgtgtttgtc gaaattccaa ggggattcag agatcaacaa gctcaagtgc ggccatttgt
     1201 ttcacaaaac atgcttggag aaatggatag actattggaa catcacttgc ccattgtgta
     1261 ggactcctct tgttgttgtg ccagaagacc atcagctttc ttctaatgtt tggtgaaacc
     1321 cagctttctt gtacaaagtg gtgatatcgc tagctagata gctagaagct tgcggccgca
     1381 cagctgtata cacgtgcaag ccagccagaa ctcgctcctg aagacccaga ggatctcgag
     1441 caccaccacc accaccacta atgttaatta agttgggcgt tcctaggctg ataaaacaga
     1501 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
     1561 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
     1621 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     1681 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
     1741 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
     1801 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt
     1861 ctaaatacat tcaaatatgt atccgctgag caataactag cataacccct tggggcctct
     1921 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1981 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2041 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2101 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2161 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2221 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2281 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2341 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2401 acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt
     2461 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2521 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2581 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2641 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2701 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2761 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2821 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2881 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2941 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3001 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3061 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3121 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3181 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3241 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3301 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3361 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3421 ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3481 aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg
     3541 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3601 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3661 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3721 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3781 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3841 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3901 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3961 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4021 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4081 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4141 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4201 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4261 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4321 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4381 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4441 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4501 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4561 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4621 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4681 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4741 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4801 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4861 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4921 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4981 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     5041 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     5101 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     5161 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     5221 ggtcctcaac gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct
     5281 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5341 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5401 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5461 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5521 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5581 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5641 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5701 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5761 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5821 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5881 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5941 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6001 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6061 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6121 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6181 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6241 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6301 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6361 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6421 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6481 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6541 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6601 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6661 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6721 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6781 ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaa
//
