LOCUS       Exported                9525 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Level 2 construct.
ACCESSION   .
VERSION     .
KEYWORDS    pUG-301
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9525)
  AUTHORS   Kowarschik K, Hoehenwarter W, Marillonnet S, Trujillo M
  TITLE     UbiGate: a synthetic biology toolbox to analyse ubiquitination.
  JOURNAL   New Phytol. 2018 Mar;217(4):1749-1763. doi: 10.1111/nph.14900. Epub 
            2017 Nov 30.
  PUBMED    29194629
REFERENCE   2  (bases 1 to 9525)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9525
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     947..966
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(986..1063)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1269..1288
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1372..1391
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1372..1390
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1391..1415
                     /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)."
     CDS             1469..1486
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5034..5120
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             5097..5123
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5136..5222
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             5199..5225
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5831..5849)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5845..5892
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5929..6384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6016..6035)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6226..6247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6476..7291)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7199..7218
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7283..7302
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7413..8001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7902..7921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8155..8172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8187..8326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8252..8271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8412..8434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8428..8619)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(9428..985)
                     /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"
ORIGIN
        1 tggtttttct tttcaccagt gacacgggca acagctgatt gcccttcacc gcctggccct
       61 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
      121 tggtggttaa cggcgggata taacatgagc tgtcctcggt atcgtcgtat cccactaccg
      181 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
      241 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
      301 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
      361 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
      421 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
      481 gtcgcgtacc gtcctcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
      541 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
      601 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
      661 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
      721 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
      781 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
      841 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
      901 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
      961 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     1021 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     1081 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     1141 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     1201 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     1261 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     1321 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     1381 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgccttaac
     1441 tttaagaagg agatatacca tgggcagcca ccaccaccac caccacagcg gaatgcagat
     1501 ctttgttaag actctcaccg gaaagacaat caccctagag gtggaaagct ccgacaccat
     1561 cgacaacgtt aaggccaaga tccaggataa ggagggcatt cctccggatc agcagaggct
     1621 tattttcgcc ggcaagcagc tagaggatgg ccgtacgttg gctgattaca atatccagaa
     1681 ggaatccacc ctccacttgg tcctcaggct ccgtggtggt taagcttgca attaacttta
     1741 agaaggagat atacaatgct tcacaagcga gctagtgaag ctaacgataa gaacgataat
     1801 accatcatcg gttccgattt agcatcatct aagaaacgcc gtatcgattt cactgaatct
     1861 tcgtctgata agtcatcttc gattttagct tctggtagta gcaggggttt tcacggcgat
     1921 agcgtcgttc agcaaatcga catggctttt ggtaattcga accgtcagga gattgatgaa
     1981 gatctgcaca gtcgccagct cgccgtctat ggtcgtgaga ctatgaggcg tctctttgct
     2041 tccaatgttc tcatctcggg gatgcacggt cttggtgccg agattgccaa gaatcttata
     2101 cttgctggtg tgaagtctgt gaccttgcat gatgaaagag ttgtagagct atgggactta
     2161 tcaagcaact ttgttttctc ggaggatgat gttggcaaga atagagcaga tgcttctgtt
     2221 cagaagttac aggatcttaa caatgctgtg gttgtctcta gcttgaccaa aagcttaaac
     2281 aaagaggatc tttctggctt ccaggttgtt gtattctctg acataagcat ggaaagagca
     2341 atcgaatttg atgactattg tcacagccat cagcctccta tcgcttttgt caaggctgat
     2401 gtcaggggtc ttttcggctc tgtcttttgt gattttgggc ctgaatttgc ggttcttgat
     2461 gttgatggag aggaacccca tacaggcatc attgcgtcta tctctaatga gaaccaggca
     2521 tttatttcct gtgtcgacga tgagaggctt gagtttgaag atggtgacct agttgttttc
     2581 tcagaagttg agggtatgac agaattgaat gatgggaaac caaggaagat aaaaagtaca
     2641 cggccatatt cattcaccct tgatgaggac actactaact atggaacata tgtgaagggt
     2701 ggtattgtca ctcaagtgaa acagccaaag ttactgaatt tcaagccatt gagggaagcg
     2761 cttaaggatc caggggattt tctatttagt gatttctcta agtttgatcg gcctccactc
     2821 cttcacttag cattccaggc acttgatcat tttaaagctg aagctggcag atttcctgtt
     2881 gctggctcag aagaggacgc tcagaagctt atatcgattg ccacagccat caataccggg
     2941 cagggtgatt taaaggtgga gaatgttgat cagaagcttc taagacactt ttcctttgga
     3001 gccaaggctg tcctgaatcc tatggctgca atgtttggcg gtattgttgg acaggaggtt
     3061 gttaaagctt gctctggaaa atttcatcct ctcttccagt ttttctactt tgattcagtg
     3121 gagtcactcc cctctgaacc tgtggactcg agtgactttg caccaagaaa cagccggtat
     3181 gatgcccaaa tatctgtatt tggggccaag ttccagaaga aacttgaaga tgctaaagtt
     3241 ttcacagtag ggtctggtgc tcttggctgt gagttcttga aaaatttggc tctgatgggg
     3301 gtttcttgtg gaagccaagg gaagctgaca gtgacagatg atgatatcat tgagaagagt
     3361 aacctcagtc gtcaatttct gttccgtgac tggaacattg gacaggctaa atccacagtt
     3421 gctgcttcgg ctgctgctgt tataaatcct cgcttcaaca ttgaggccct gcaaaaccgt
     3481 gttggcgcgg agactgagaa tgtatttgac gatgccttct gggagaactt aactgttgtc
     3541 gtcaatgcac tggataatgt taatgcgagg ctctatgttg attcgaggtg cttgtatttc
     3601 cagaagcctc tccttgagtc tggaactctt ggtacaaagt gcaacacaca gtcggttatc
     3661 ccacatctaa ctgaaaatta cggtgcctca agggacccac cagagaaaca ggcccccatg
     3721 tgtacagtgc attccttccc gcataacatt gatcactgtt taacttgggc tcgctctgag
     3781 tttgagggtc tgctggagaa aactcccgct gaagtgaatg catatctttc tagcccagtt
     3841 gagtacacta actcaatgat gagtgctggt gatgctcagg cgagggacac attggagagg
     3901 attgttgagt gccttgaaaa ggagaagtgt gaaaccttcc aagactgctt aacctgggca
     3961 cgactgaggt ttgaggatta ctttgtgaac cgtgtcaagc aattgatata cacatttcct
     4021 gaagatgctg cgactagtac cggagctcca ttctggtctg ctccaaaaag attccctcgt
     4081 ccactccagt actcgtcgtc cgacccaagt ctccttaact ttatcacagc gactgctatt
     4141 ttacgagcag agacatttgg gatcccgata cctgagtgga ctaagaaccc aaaggaagca
     4201 gcagaagctg ttgacagagt gatagtccca gactttgagc caaggcaaga tgcaaagatt
     4261 gtgactgatg agaaagctac cactttaacc actgcttcgg tggacgacgc tgcagtcatc
     4321 gacgatctca ttgctaagat cgaccagtgt aggcataact tgtcgccaga cttcaggatg
     4381 aaacctattc agttcgaaaa ggatgatgat acaaactatc acatggatgt gatagcagga
     4441 ctagccaaca tgagggcgag aaattacagc atccctgaag tggacaagct gaaagcaaag
     4501 ttcatagcag ggagaatcat cccagccatt gcgacttcaa cagccatggc tactgggctg
     4561 gtctgcctcg agctgtacaa ggtccttgat ggaggacata aagtggaagc ttacagaaac
     4621 acatttgcca acctggcact tccactattc tccatggctg aaccacttcc accaaaggtg
     4681 gtgaagcacc gtgacatggc ttggacagtt tgggacagat gggttctcaa gggaaaccca
     4741 actcttcgtg aggtgttgca gtggctggag gacaaagggc ttagcgcata cagcatctct
     4801 tgtggaagct gtctactgtt caacagtatg ttcacaaggc acaaggagag gatggacaag
     4861 aaggttgtgg atctcgctcg ggatgttgcg aaagtcgagt taccgcctta tcgcaaccat
     4921 cttgatgtag tggtggcttg tgaggatgaa gatgacaatg acgtcgatat tcctctcgtc
     4981 tctatctact tcaggtgagc ttactattaa ctttaagaag gagatatacc atgtacccat
     5041 acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca ggattgtatc
     5101 catatgacgt tccagattac gccactagag ctgcttaccc atacgatgtt cctgactatg
     5161 cgggctatcc ctatgacgtc ccggactatg caggattgta tccatatgac gttccagatt
     5221 acgcaatggc gtcgaagcgg atcttgaagg aattgaagga tctccagaag gatccaccta
     5281 catcatgtag cgcaggtcct gttgctgagg acatgtttca ctggcaggca acgataatgg
     5341 gtccttcaga gagtccttat gctggaggtg ttttccttgt aaccatccat ttccctccgg
     5401 attacccatt caagcctcct aaggtggcct ttaggaccaa ggtgttccat cccaacatta
     5461 acagcaacgg tagcatttgc ctcgacatct tgaaggagca gtggagtcct gcgctcacca
     5521 tttccaaggt gctgctatcg atctgttctt tgttaaccga tccaaacccg gatgatcctt
     5581 tggtgcccga gatagctcac atgtacaaga ctgacaagaa caagtacgag tccactgcac
     5641 gaagctggac tcagaagtat gccatgggct aagcttttac gcttttaagc cagccccgac
     5701 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5761 gacaagctgt gaccgggatg agatccggct gctaacaaag cccgaaagga agctgagttg
     5821 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     5881 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     5941 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     6001 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     6061 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     6121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     6181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     6241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     6301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     6361 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     6481 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     6541 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     6601 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     6661 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     6721 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     6781 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     6841 gagcgagtcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     6901 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     6961 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     7021 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     7081 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     7141 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     7201 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     7261 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     7321 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7381 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7441 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7501 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     7561 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7621 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7681 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7741 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7801 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7861 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7921 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7981 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8041 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     8101 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     8161 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     8221 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     8281 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     8341 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     8401 tgtgaccgac tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     8461 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     8521 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     8581 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     8641 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     8701 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     8761 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     8821 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     8881 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccca agaccattca
     8941 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     9001 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     9061 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     9121 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     9181 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     9241 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga acacagtcat
     9301 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     9361 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     9421 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     9481 cggccaacgc gcggggagag gcggtttgcg tattgggcgc caggg
//
