LOCUS       Exported                9564 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Level 2 construct.
ACCESSION   .
VERSION     .
KEYWORDS    pUG-308
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9564)
  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 9564)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9564
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     568..587
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(607..684)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     890..909
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     993..1012
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        993..1011
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1012..1036
                     /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             1090..1107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4655..4741
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             4718..4744
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4757..4843
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             4820..4846
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5491..5509)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5505..5552
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5589..6044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5676..5695)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5886..5907
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6136..6951)
                     /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     6859..6878
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6943..6962
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7073..7661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7562..7581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7815..7832
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7847..7986
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7912..7931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8072..8094
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8088..8279)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(9088..606)
                     /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 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
       61 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcctcatg
      121 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      181 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      241 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      301 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      361 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      421 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      481 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      541 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      601 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      661 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
      721 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
      781 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
      841 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
      901 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
      961 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     1021 agcggataac aattcccctc tagaaataat tttgccttaa ctttaagaag gagatatacc
     1081 atgggcagcc accaccacca ccaccacagc ggaatgcaga tctttgttaa gactctcacc
     1141 ggaaagacaa tcaccctaga ggtggaaagc tccgacacca tcgacaacgt taaggccaag
     1201 atccaggata aggagggcat tcctccggat cagcagaggc ttattttcgc cggcaagcag
     1261 ctagaggatg gccgtacgtt ggctgattac aatatccaga aggaatccac cctccacttg
     1321 gtcctcaggc tccgtggtgg ttaagcttgc aattaacttt aagaaggaga tatacaatgc
     1381 ttcacaagcg agctagtgaa gctaacgata agaacgataa taccatcatc ggttccgatt
     1441 tagcatcatc taagaaacgc cgtatcgatt tcactgaatc ttcgtctgat aagtcatctt
     1501 cgattttagc ttctggtagt agcaggggtt ttcacggcga tagcgtcgtt cagcaaatcg
     1561 acatggcttt tggtaattcg aaccgtcagg agattgatga agatctgcac agtcgccagc
     1621 tcgccgtcta tggtcgtgag actatgaggc gtctctttgc ttccaatgtt ctcatctcgg
     1681 ggatgcacgg tcttggtgcc gagattgcca agaatcttat acttgctggt gtgaagtctg
     1741 tgaccttgca tgatgaaaga gttgtagagc tatgggactt atcaagcaac tttgttttct
     1801 cggaggatga tgttggcaag aatagagcag atgcttctgt tcagaagtta caggatctta
     1861 acaatgctgt ggttgtctct agcttgacca aaagcttaaa caaagaggat ctttctggct
     1921 tccaggttgt tgtattctct gacataagca tggaaagagc aatcgaattt gatgactatt
     1981 gtcacagcca tcagcctcct atcgcttttg tcaaggctga tgtcaggggt cttttcggct
     2041 ctgtcttttg tgattttggg cctgaatttg cggttcttga tgttgatgga gaggaacccc
     2101 atacaggcat cattgcgtct atctctaatg agaaccaggc atttatttcc tgtgtcgacg
     2161 atgagaggct tgagtttgaa gatggtgacc tagttgtttt ctcagaagtt gagggtatga
     2221 cagaattgaa tgatgggaaa ccaaggaaga taaaaagtac acggccatat tcattcaccc
     2281 ttgatgagga cactactaac tatggaacat atgtgaaggg tggtattgtc actcaagtga
     2341 aacagccaaa gttactgaat ttcaagccat tgagggaagc gcttaaggat ccaggggatt
     2401 ttctatttag tgatttctct aagtttgatc ggcctccact ccttcactta gcattccagg
     2461 cacttgatca ttttaaagct gaagctggca gatttcctgt tgctggctca gaagaggacg
     2521 ctcagaagct tatatcgatt gccacagcca tcaataccgg gcagggtgat ttaaaggtgg
     2581 agaatgttga tcagaagctt ctaagacact tttcctttgg agccaaggct gtcctgaatc
     2641 ctatggctgc aatgtttggc ggtattgttg gacaggaggt tgttaaagct tgctctggaa
     2701 aatttcatcc tctcttccag tttttctact ttgattcagt ggagtcactc ccctctgaac
     2761 ctgtggactc gagtgacttt gcaccaagaa acagccggta tgatgcccaa atatctgtat
     2821 ttggggccaa gttccagaag aaacttgaag atgctaaagt tttcacagta gggtctggtg
     2881 ctcttggctg tgagttcttg aaaaatttgg ctctgatggg ggtttcttgt ggaagccaag
     2941 ggaagctgac agtgacagat gatgatatca ttgagaagag taacctcagt cgtcaatttc
     3001 tgttccgtga ctggaacatt ggacaggcta aatccacagt tgctgcttcg gctgctgctg
     3061 ttataaatcc tcgcttcaac attgaggccc tgcaaaaccg tgttggcgcg gagactgaga
     3121 atgtatttga cgatgccttc tgggagaact taactgttgt cgtcaatgca ctggataatg
     3181 ttaatgcgag gctctatgtt gattcgaggt gcttgtattt ccagaagcct ctccttgagt
     3241 ctggaactct tggtacaaag tgcaacacac agtcggttat cccacatcta actgaaaatt
     3301 acggtgcctc aagggaccca ccagagaaac aggcccccat gtgtacagtg cattccttcc
     3361 cgcataacat tgatcactgt ttaacttggg ctcgctctga gtttgagggt ctgctggaga
     3421 aaactcccgc tgaagtgaat gcatatcttt ctagcccagt tgagtacact aactcaatga
     3481 tgagtgctgg tgatgctcag gcgagggaca cattggagag gattgttgag tgccttgaaa
     3541 aggagaagtg tgaaaccttc caagactgct taacctgggc acgactgagg tttgaggatt
     3601 actttgtgaa ccgtgtcaag caattgatat acacatttcc tgaagatgct gcgactagta
     3661 ccggagctcc attctggtct gctccaaaaa gattccctcg tccactccag tactcgtcgt
     3721 ccgacccaag tctccttaac tttatcacag cgactgctat tttacgagca gagacatttg
     3781 ggatcccgat acctgagtgg actaagaacc caaaggaagc agcagaagct gttgacagag
     3841 tgatagtccc agactttgag ccaaggcaag atgcaaagat tgtgactgat gagaaagcta
     3901 ccactttaac cactgcttcg gtggacgacg ctgcagtcat cgacgatctc attgctaaga
     3961 tcgaccagtg taggcataac ttgtcgccag acttcaggat gaaacctatt cagttcgaaa
     4021 aggatgatga tacaaactat cacatggatg tgatagcagg actagccaac atgagggcga
     4081 gaaattacag catccctgaa gtggacaagc tgaaagcaaa gttcatagca gggagaatca
     4141 tcccagccat tgcgacttca acagccatgg ctactgggct ggtctgcctc gagctgtaca
     4201 aggtccttga tggaggacat aaagtggaag cttacagaaa cacatttgcc aacctggcac
     4261 ttccactatt ctccatggct gaaccacttc caccaaaggt ggtgaagcac cgtgacatgg
     4321 cttggacagt ttgggacaga tgggttctca agggaaaccc aactcttcgt gaggtgttgc
     4381 agtggctgga ggacaaaggg cttagcgcat acagcatctc ttgtggaagc tgtctactgt
     4441 tcaacagtat gttcacaagg cacaaggaga ggatggacaa gaaggttgtg gatctcgctc
     4501 gggatgttgc gaaagtcgag ttaccgcctt atcgcaacca tcttgatgta gtggtggctt
     4561 gtgaggatga agatgacaat gacgtcgata ttcctctcgt ctctatctac ttcaggtgag
     4621 cttactatta actttaagaa ggagatatac catgtaccca tacgatgttc ctgactatgc
     4681 gggctatccc tatgacgtcc cggactatgc aggattgtat ccatatgacg ttccagatta
     4741 cgccactaga gctgcttacc catacgatgt tcctgactat gcgggctatc cctatgacgt
     4801 cccggactat gcaggattgt atccatatga cgttccagat tacgcaatga cgagttcttc
     4861 tgaatcaact cgcaagggtc tgaccaaaat cgccacgaac cggttgcaga aagagttcat
     4921 ggagtggcaa accaatcctc cttctggttt caagcacaga gtctctgata atctccaacg
     4981 atggatcatt gaagtgcatg gagttccagg aactctttac gccaatgaaa cttaccaact
     5041 tcaagttgag ttccctgagc attatcctat ggaagctcca caggttatct ttcagcatcc
     5101 agctccactt catcctcata tttacagcaa cggtcacatt tgtttagatg tcttgtatga
     5161 ttcgtggtca ccggcgatga gactaagttc catctgtctc agcattctct caatgctctc
     5221 cagctcatcc gtgaagcaaa agccgaaaga caatgaccat tacttgaaaa actgcaaaca
     5281 tggaagatct cccaaagaaa ctaggtggcg tttccatgac gataaagtat aagcttttac
     5341 gcttttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5401 gctcccggca tccgcttaca gacaagctgt gaccgggatg agatccggct gctaacaaag
     5461 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     5521 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     5581 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5641 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5701 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5761 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5821 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5881 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5941 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     6001 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     6061 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6121 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     6181 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     6241 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     6301 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     6361 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     6421 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     6481 attcattcgt gattgcgcct gagcgagtcg aaatacgcga tcgctgttaa aaggacaatt
     6541 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     6601 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     6661 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     6721 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     6781 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     6841 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     6901 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     6961 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     7021 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     7081 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     7141 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7201 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7261 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7321 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7381 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7441 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7501 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7561 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7621 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7681 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7741 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7801 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     7861 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     7921 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     7981 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     8041 gcatccgctt acagacaagc tgtgaccgac tccgggagct gcatgtgtca gaggttttca
     8101 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     8161 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     8221 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     8281 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     8341 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     8401 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     8461 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     8521 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     8581 cggaaaccca agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     8641 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     8701 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     8761 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     8821 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     8881 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     8941 atgataaaga acacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     9001 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     9061 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     9121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     9181 cagggtggtt tttcttttca ccagtgacac gggcaacagc tgattgccct tcaccgcctg
     9241 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     9301 tttgatggtg gttaacggcg ggatataaca tgagctgtcc tcggtatcgt cgtatcccac
     9361 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     9421 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     9481 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     9541 aatttgattg cgagtgagat attt
//
