LOCUS       sequence-229862-        7558 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 7558)
  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 7558)
  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 544-XERICO
FEATURES             Location/Qualifiers
     source          1..7558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        22..40
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    41..65
                     /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             80..102
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             119..136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             140..1624
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV
                     RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR
                     ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL
                     REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE
                     IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA
                     QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT
                     VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD
                     EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED
                     LAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     protein_bind    1634..1658
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2151..2175)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2185..2202
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2368..2454
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2546..2573
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2627..2645)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2641..2688
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2725..3180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2812..2831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3022..3043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3295..4086,4087..4155))
                     /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     3918..3937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4156..4248)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4329..4917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4818..4837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5071..5088
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5103..5242
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5168..5187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5328..5350
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5344..5535)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6107..7189)
                     /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     7151..7170
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7190..7267)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7473..7492
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
       61 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgggatccca
      121 tcaccatcac catcacagta tgaacaaaga aattttggct gtagttgaag ccgtatccaa
      181 tgaaaaggcg ctacctcgcg agaagatttt cgaagcattg gaaagcgcgc tggcgacagc
      241 aacaaagaaa aaatatgaac aagagatcga cgtccgcgta cagatcgatc gcaaaagcgg
      301 tgattttgac actttccgtc gctggttagt tgttgatgaa gtcacccagc cgaccaagga
      361 aatcaccctt gaagccgcac gttatgaaga tgaaagcctg aacctgggcg attacgttga
      421 agatcagatt gagtctgtta cctttgaccg tatcactacc cagacggcaa aacaggttat
      481 cgtgcagaaa gtgcgtgaag ccgaacgtgc gatggtggtt gatcagttcc gtgaacacga
      541 aggtgaaatc atcaccggcg tggtgaaaaa agtaaaccgc gacaacatct ctctggatct
      601 gggcaacaac gctgaagccg tgatcctgcg cgaagatatg ctgccgcgtg aaaacttccg
      661 ccctggcgac cgcgttcgtg gcgtgctcta ttccgttcgc ccggaagcgc gtggcgcgca
      721 actgttcgtc actcgttcca agccggaaat gctgatcgaa ctgttccgta ttgaagtgcc
      781 agaaatcggc gaagaagtga ttgaaattaa agcagcggct cgcgatccgg gttctcgtgc
      841 gaaaatcgcg gtgaaaacca acgataaacg tatcgatccg gtaggtgctt gcgtaggtat
      901 gcgtggcgcg cgtgttcagg cggtgtctac tgaactgggt ggcgagcgta tcgatatcgt
      961 cctgtgggat gataacccgg cgcagttcgt gattaacgca atggcaccgg cagacgttgc
     1021 ttctatcgtg gtggatgaag ataaacacac catggacatc gccgttgaag ccggtaatct
     1081 ggcgcaggcg attggccgta acggtcagaa cgtgcgtctg gcttcgcaac tgagcggttg
     1141 ggaactcaac gtgatgaccg ttgacgacct gcaagctaag catcaggcgg aagcgcacgc
     1201 agcgatcgac accttcacca aatatctcga catcgacgaa gacttcgcga ctgttctggt
     1261 agaagaaggc ttctcgacgc tggaagaatt ggcctatgtg ccgatgaaag agctgttgga
     1321 aatcgaaggc cttgatgagc cgaccgttga agcactgcgc gagcgtgcta aaaatgcact
     1381 ggccaccatt gcacaggccc aggaagaaag cctcggtgat aacaaaccgg ctgacgatct
     1441 gctgaacctt gaaggggtag atcgtgattt ggcattcaaa ctggccgccc gtggcgtttg
     1501 tacgctggaa gatctcgccg aacagggcat tgatgatctg gctgatatcg aagggttgac
     1561 cgacgaaaaa gccggagcac tgattatggc tgcccgtaat atttgctggt tcggtgacga
     1621 agcgactaga tcaacaagtt tgtacaaaaa agcaggctct atgggtctat caagtcttcc
     1681 tggtccatca gaaggaatgt tatgtgtgat attagttaat acagcattat cgatctccat
     1741 tgtcaaaggc attgtaagat cattccttgg catagtagga atcagtctct cgccgtcttc
     1801 atcctcgcct tcttcggtga cggtatcttc agagaattca tcaacttcag agtcatttga
     1861 tttccgggtc tgccaaccag agagttacct tgaggagttc aggaaccgga ctccgacact
     1921 gaggtttgag agcttgtgca ggtgcaagaa acaggcagac aatgagtgtt ctgtgtgttt
     1981 gtcgaaattc caaggggatt cagagatcaa caagctcaag tgcggccatt tgtttcacaa
     2041 aacatgcttg gagaaatgga tagactattg gaacatcact tgcccattgt gtaggactcc
     2101 tcttgttgtt gtgccagaag accatcagct ttcttctaat gtttggtgaa acccagcttt
     2161 cttgtacaaa gtggttgatt cgagcaccac caccaccacc actaatgtta attaagttgg
     2221 gcgttcctag gctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
     2281 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc
     2341 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     2401 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     2461 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     2521 taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt
     2581 ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc tgagcaataa
     2641 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2701 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2761 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2821 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2881 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2941 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3001 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3061 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3121 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     3181 ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3241 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3301 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3361 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3421 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3481 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3541 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3601 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3661 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3721 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3781 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3841 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3901 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3961 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4021 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4081 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4141 atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat cagggttatt
     4201 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggtcatgacc
     4261 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4321 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4381 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4441 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4501 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4561 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4621 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4681 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4741 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4801 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4861 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4921 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4981 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5041 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5101 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     5161 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     5221 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     5281 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5341 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5401 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5461 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5521 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5581 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5641 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5701 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5761 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5821 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5881 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5941 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat
     6001 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     6061 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     6121 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6181 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     6241 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     6301 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     6361 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     6421 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     6481 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     6541 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6601 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     6661 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     6721 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     6781 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     6841 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     6901 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6961 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     7021 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     7081 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     7141 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     7201 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     7261 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     7321 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     7381 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     7441 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     7501 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgaga
//
