LOCUS       sequence-229856-        7668 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 7668)
  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 7668)
  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 566-PHL2
FEATURES             Location/Qualifiers
     source          1..7668
                     /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             120..1217
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1074..1083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1191..1214
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1236..1260
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2152..2176)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2236..2268
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2275..2292
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2458..2544
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2636..2663
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2717..2735)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2731..2778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2815..3270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2902..2921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3112..3133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3385..4176,4177..4245))
                     /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     4008..4027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4246..4338)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4419..5007
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4908..4927
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5161..5178
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5193..5332
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5258..5277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5418..5440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5434..5625)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6197..7279)
                     /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     7241..7260
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7280..7357)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7563..7582
                     /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 catcacggca
      121 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      181 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      241 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
      301 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
      361 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
      421 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
      481 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
      541 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
      601 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
      661 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
      721 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
      781 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
      841 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
      901 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
      961 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1021 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca
     1081 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1141 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1201 tgaaagacgc gcagactaat tcgggatctg atatcacaag tttgtacaaa aaagcaggct
     1261 ctatgtactc agcgattcgc tcgcttccac tcgatggtgg acacgttggt ggtgactacc
     1321 atggacctct tgacggaacc aatcttcccg gtgacgcttg tttggtttta acgactgacc
     1381 ctaaacctcg tctccggtgg acaactgagc ttcatgagag attcgttgac gccgttactc
     1441 agctcggtgg tcctgacaaa gcgactccca aaactattat gagaacaatg ggagtgaagg
     1501 gtctcactct ctaccacctc aaatcacatc ttcagaaatt ccgcctaggg aggcaagctg
     1561 gcaaagaatc aactgagaac tctaaagatg cttcttgtgt aggggagagt caggacacag
     1621 gttcatcttc gacatcatca atgagaatgg cgcagcagga gcagaacgag ggttaccaag
     1681 tcaccgaagc tctacgtgct cagatggaag tccaaagaag actacacgat caattggagg
     1741 tgcaacggag gctccagctg aggatagagg cacaaggaaa atacctgcaa tcgattcttg
     1801 aaaaagcttg caaggccttt gacgagcaag ctgctacttt tgctggactt gaggctgcta
     1861 gggaagagct atcagagcta gccatcaaag tctccaatag ctctcaagga acatcagtcc
     1921 cgtacttcga tgcaacaaag atgatgatga tgccatcgtt gtcagagctt gcagtagcaa
     1981 tagacaacaa aaacaacatc acaaccaact gttcagtaga aagctctctg acttccatca
     2041 cacatgggag ctctatatct gctgcatcaa tgaagaagcg tcaacgtgga gacaatttgg
     2101 gcgtagggta tgaatcaggc tggattatgc ctagtagcac cattggataa aacccagctt
     2161 tcttgtacaa agtggtgata tcgctagcta gatagctaga agcttgcggc cgcacagctg
     2221 tatacacgtg caagccagcc agaactcgct cctgaagacc cagaggatct cgagcaccac
     2281 caccaccacc actaatgtta attaagttgg gcgttcctag gctgataaaa cagaatttgc
     2341 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     2401 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     2461 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     2521 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     2581 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     2641 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat
     2701 acattcaaat atgtatccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2761 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     2821 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2881 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2941 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3001 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3061 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3121 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3181 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3241 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     3301 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3361 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3421 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3481 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3541 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3601 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3661 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3721 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3781 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3841 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3901 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3961 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4021 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4081 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4141 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4201 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4261 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4321 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4381 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4441 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4501 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4561 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4621 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4681 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4741 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4801 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4861 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4921 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4981 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5041 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5101 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5161 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5221 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5281 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5341 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5401 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5461 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5521 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5581 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5641 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5701 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5761 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     5821 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     5881 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     5941 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6001 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6061 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     6121 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     6181 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6241 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     6301 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6361 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6421 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     6481 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6541 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6601 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6661 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6721 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6781 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6841 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6901 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6961 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     7021 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     7081 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     7141 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     7201 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     7261 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     7321 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     7381 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     7441 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     7501 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     7561 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     7621 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaa
//
