LOCUS       sequence-229730-        6567 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 6567)
  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 6567)
  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 527-PHL2
FEATURES             Location/Qualifiers
     source          1..6567
                     /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             105..122
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    135..159
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1051..1075)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1135..1167
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1174..1191
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1357..1443
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1535..1562
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1616..1634)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1630..1677
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1714..2169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1801..1820)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2011..2032
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2284..3075,3076..3144))
                     /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     2907..2926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3145..3237)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3318..3906
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3807..3826
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4060..4077
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4092..4231
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4157..4176)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4317..4339
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4333..4524)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5096..6178)
                     /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     6140..6159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6179..6256)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6462..6481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatctgg atcccaccat caccatcacc
      121 atagatctga tatcacaagt ttgtacaaaa aagcaggctc tatgtactca gcgattcgct
      181 cgcttccact cgatggtgga cacgttggtg gtgactacca tggacctctt gacggaacca
      241 atcttcccgg tgacgcttgt ttggttttaa cgactgaccc taaacctcgt ctccggtgga
      301 caactgagct tcatgagaga ttcgttgacg ccgttactca gctcggtggt cctgacaaag
      361 cgactcccaa aactattatg agaacaatgg gagtgaaggg tctcactctc taccacctca
      421 aatcacatct tcagaaattc cgcctaggga ggcaagctgg caaagaatca actgagaact
      481 ctaaagatgc ttcttgtgta ggggagagtc aggacacagg ttcatcttcg acatcatcaa
      541 tgagaatggc gcagcaggag cagaacgagg gttaccaagt caccgaagct ctacgtgctc
      601 agatggaagt ccaaagaaga ctacacgatc aattggaggt gcaacggagg ctccagctga
      661 ggatagaggc acaaggaaaa tacctgcaat cgattcttga aaaagcttgc aaggcctttg
      721 acgagcaagc tgctactttt gctggacttg aggctgctag ggaagagcta tcagagctag
      781 ccatcaaagt ctccaatagc tctcaaggaa catcagtccc gtacttcgat gcaacaaaga
      841 tgatgatgat gccatcgttg tcagagcttg cagtagcaat agacaacaaa aacaacatca
      901 caaccaactg ttcagtagaa agctctctga cttccatcac acatgggagc tctatatctg
      961 ctgcatcaat gaagaagcgt caacgtggag acaatttggg cgtagggtat gaatcaggct
     1021 ggattatgcc tagtagcacc attggataaa acccagcttt cttgtacaaa gtggtgatat
     1081 cgctagctag atagctagaa gcttgcggcc gcacagctgt atacacgtgc aagccagcca
     1141 gaactcgctc ctgaagaccc agaggatctc gagcaccacc accaccacca ctaatgttaa
     1201 ttaagttggg cgttcctagg ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     1261 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     1321 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     1381 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     1441 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     1501 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     1561 tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct
     1621 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1681 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1741 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1801 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1861 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1921 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1981 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2041 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2101 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     2161 ttacaatttc tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct
     2221 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2281 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2341 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2401 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2461 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2521 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2581 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2641 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2701 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2761 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2821 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2881 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2941 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3001 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3061 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3121 gacacggaaa tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc
     3181 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3241 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3301 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3361 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3421 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3481 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3541 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3601 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3661 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3721 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3781 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3841 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3901 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3961 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4021 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4081 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4141 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4201 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4261 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4321 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4381 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4441 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4501 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4561 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4621 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4681 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4741 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4801 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4861 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4921 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4981 gctagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5041 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5101 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5161 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5221 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5281 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5341 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     5401 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5461 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5521 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5581 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5641 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5701 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5761 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5821 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5881 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5941 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6001 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6061 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6121 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6181 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6241 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     6301 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     6361 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     6421 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     6481 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     6541 atcgagatcg atctcgatcc cgcgaaa
//
