LOCUS       sequence-229854-        7233 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 7233)
  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 7233)
  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-PHL2
FEATURES             Location/Qualifiers
     source          1..7233
                     /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(1717..1741)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1801..1833
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1840..1857
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2023..2109
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2201..2228
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2282..2300)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2296..2343
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2380..2835
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2467..2486)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2677..2698
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2950..3741,3742..3810))
                     /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     3573..3592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3811..3903)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3984..4572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4473..4492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4726..4743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4758..4897
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4823..4842)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4983..5005
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4999..5190)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5762..6844)
                     /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     6806..6825
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6845..6922)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7128..7147
                     /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 tactcagcga
      841 ttcgctcgct tccactcgat ggtggacacg ttggtggtga ctaccatgga cctcttgacg
      901 gaaccaatct tcccggtgac gcttgtttgg ttttaacgac tgaccctaaa cctcgtctcc
      961 ggtggacaac tgagcttcat gagagattcg ttgacgccgt tactcagctc ggtggtcctg
     1021 acaaagcgac tcccaaaact attatgagaa caatgggagt gaagggtctc actctctacc
     1081 acctcaaatc acatcttcag aaattccgcc tagggaggca agctggcaaa gaatcaactg
     1141 agaactctaa agatgcttct tgtgtagggg agagtcagga cacaggttca tcttcgacat
     1201 catcaatgag aatggcgcag caggagcaga acgagggtta ccaagtcacc gaagctctac
     1261 gtgctcagat ggaagtccaa agaagactac acgatcaatt ggaggtgcaa cggaggctcc
     1321 agctgaggat agaggcacaa ggaaaatacc tgcaatcgat tcttgaaaaa gcttgcaagg
     1381 cctttgacga gcaagctgct acttttgctg gacttgaggc tgctagggaa gagctatcag
     1441 agctagccat caaagtctcc aatagctctc aaggaacatc agtcccgtac ttcgatgcaa
     1501 caaagatgat gatgatgcca tcgttgtcag agcttgcagt agcaatagac aacaaaaaca
     1561 acatcacaac caactgttca gtagaaagct ctctgacttc catcacacat gggagctcta
     1621 tatctgctgc atcaatgaag aagcgtcaac gtggagacaa tttgggcgta gggtatgaat
     1681 caggctggat tatgcctagt agcaccattg gataaaaccc agctttcttg tacaaagtgg
     1741 tgatatcgct agctagatag ctagaagctt gcggccgcac agctgtatac acgtgcaagc
     1801 cagccagaac tcgctcctga agacccagag gatctcgagc accaccacca ccaccactaa
     1861 tgttaattaa gttgggcgtt cctaggctga taaaacagaa tttgcctggc ggcagtagcg
     1921 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     1981 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     2041 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2101 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     2161 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     2221 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     2281 tccgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     2341 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     2401 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2461 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2521 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2581 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2641 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2701 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2761 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2821 taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta
     2881 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2941 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3001 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3061 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3121 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3181 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3241 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3301 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3361 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3421 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3481 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3541 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3601 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3661 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3721 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3781 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatcat gattgaagca
     3841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3901 aaataggtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3961 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4021 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4081 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4141 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4201 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4261 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4321 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4381 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4441 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4501 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4561 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4621 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4681 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4741 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4801 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4861 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4921 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4981 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5041 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5101 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5161 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5221 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5281 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5341 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5401 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5461 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5521 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5581 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5641 gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5701 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5761 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5821 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5881 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5941 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6001 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     6061 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6121 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6181 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6241 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6301 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6361 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6421 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6481 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6541 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6601 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6661 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6721 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6781 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6841 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6901 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6961 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     7021 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     7081 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     7141 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     7201 tagaggatcg agatcgatct cgatcccgcg aaa
//
