LOCUS       sequence-229700-        7957 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 7957)
  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 7957)
  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-PHL2
FEATURES             Location/Qualifiers
     source          1..7957
                     /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(2550..2574)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2584..2601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2767..2853
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2945..2972
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3026..3044)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3040..3087
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3124..3579
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3211..3230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3421..3442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3694..4485,4486..4554))
                     /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     4317..4336
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4555..4647)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4728..5316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5217..5236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5470..5487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5502..5641
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5567..5586)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5727..5749
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5743..5934)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6506..7588)
                     /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     7550..7569
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7589..7666)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7872..7891
                     /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 atgtactcag cgattcgctc
     1681 gcttccactc gatggtggac acgttggtgg tgactaccat ggacctcttg acggaaccaa
     1741 tcttcccggt gacgcttgtt tggttttaac gactgaccct aaacctcgtc tccggtggac
     1801 aactgagctt catgagagat tcgttgacgc cgttactcag ctcggtggtc ctgacaaagc
     1861 gactcccaaa actattatga gaacaatggg agtgaagggt ctcactctct accacctcaa
     1921 atcacatctt cagaaattcc gcctagggag gcaagctggc aaagaatcaa ctgagaactc
     1981 taaagatgct tcttgtgtag gggagagtca ggacacaggt tcatcttcga catcatcaat
     2041 gagaatggcg cagcaggagc agaacgaggg ttaccaagtc accgaagctc tacgtgctca
     2101 gatggaagtc caaagaagac tacacgatca attggaggtg caacggaggc tccagctgag
     2161 gatagaggca caaggaaaat acctgcaatc gattcttgaa aaagcttgca aggcctttga
     2221 cgagcaagct gctacttttg ctggacttga ggctgctagg gaagagctat cagagctagc
     2281 catcaaagtc tccaatagct ctcaaggaac atcagtcccg tacttcgatg caacaaagat
     2341 gatgatgatg ccatcgttgt cagagcttgc agtagcaata gacaacaaaa acaacatcac
     2401 aaccaactgt tcagtagaaa gctctctgac ttccatcaca catgggagct ctatatctgc
     2461 tgcatcaatg aagaagcgtc aacgtggaga caatttgggc gtagggtatg aatcaggctg
     2521 gattatgcct agtagcacca ttggataaaa cccagctttc ttgtacaaag tggttgattc
     2581 gagcaccacc accaccacca ctaatgttaa ttaagttggg cgttcctagg ctgataaaac
     2641 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     2701 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     2761 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2821 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     2881 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     2941 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     3001 tttctaaata cattcaaata tgtatccgct gagcaataac tagcataacc ccttggggcc
     3061 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     3121 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3181 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3241 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3301 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3361 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3421 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3481 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3541 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag
     3601 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3661 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3721 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3781 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3841 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3901 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3961 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4021 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4081 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4141 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4201 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4261 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4321 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4381 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4441 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4501 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4561 cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat
     4621 ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt
     4681 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4741 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4801 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4861 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4921 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4981 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5041 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5101 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5161 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5221 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5281 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5341 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5401 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5461 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5521 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     5581 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     5641 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5701 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5761 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     5821 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5881 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5941 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     6001 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     6061 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     6121 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     6181 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     6241 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     6301 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     6361 ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga
     6421 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6481 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6541 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6601 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     6661 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     6721 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     6781 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     6841 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     6901 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     6961 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     7021 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     7081 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     7141 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     7201 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     7261 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     7321 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     7381 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     7441 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     7501 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     7561 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     7621 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     7681 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     7741 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     7801 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     7861 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     7921 atgccggcca cgatgcgtcc ggcgtagagg atcgaga
//
