LOCUS       Exported                9525 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Level 2 construct.
ACCESSION   .
VERSION     .
KEYWORDS    pUG-319
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     29..48
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     132..151
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        132..150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    151..175
                     /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             229..246
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3794..3880
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             3857..3883
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3896..3982
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             3959..3985
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4591..4609)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4605..4652
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4689..5144
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4776..4795)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4986..5007
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5236..6051)
                     /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     5959..5978
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6043..6062
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6173..6761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6662..6681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6915..6932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6947..7086
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7012..7031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7172..7194
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7188..7379)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(8188..9270)
                     /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     9232..9251
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9271..9348)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
       61 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
      121 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
      181 agaaataatt ttgccttaac tttaagaagg agatatacca tgggcagcca ccaccaccac
      241 caccacagcg gaatgcagat ctttgttaag actctcaccg gaaagacaat caccctagag
      301 gtggaaagct ccgacaccat cgacaacgtt aaggccaaga tccaggataa ggagggcatt
      361 cctccggatc agcagaggct tattttcgcc ggcaagcagc tagaggatgg ccgtacgttg
      421 gctgattaca atatccagaa ggaatccacc ctccacttgg tcctcaggct ccgtggtggt
      481 taagcttgca attaacttta agaaggagat atacaatgct tcacaagcga gctagtgaag
      541 ctaacgataa gaacgataat accatcatcg gttccgattt agcatcatct aagaaacgcc
      601 gtatcgattt cactgaatct tcgtctgata agtcatcttc gattttagct tctggtagta
      661 gcaggggttt tcacggcgat agcgtcgttc agcaaatcga catggctttt ggtaattcga
      721 accgtcagga gattgatgaa gatctgcaca gtcgccagct cgccgtctat ggtcgtgaga
      781 ctatgaggcg tctctttgct tccaatgttc tcatctcggg gatgcacggt cttggtgccg
      841 agattgccaa gaatcttata cttgctggtg tgaagtctgt gaccttgcat gatgaaagag
      901 ttgtagagct atgggactta tcaagcaact ttgttttctc ggaggatgat gttggcaaga
      961 atagagcaga tgcttctgtt cagaagttac aggatcttaa caatgctgtg gttgtctcta
     1021 gcttgaccaa aagcttaaac aaagaggatc tttctggctt ccaggttgtt gtattctctg
     1081 acataagcat ggaaagagca atcgaatttg atgactattg tcacagccat cagcctccta
     1141 tcgcttttgt caaggctgat gtcaggggtc ttttcggctc tgtcttttgt gattttgggc
     1201 ctgaatttgc ggttcttgat gttgatggag aggaacccca tacaggcatc attgcgtcta
     1261 tctctaatga gaaccaggca tttatttcct gtgtcgacga tgagaggctt gagtttgaag
     1321 atggtgacct agttgttttc tcagaagttg agggtatgac agaattgaat gatgggaaac
     1381 caaggaagat aaaaagtaca cggccatatt cattcaccct tgatgaggac actactaact
     1441 atggaacata tgtgaagggt ggtattgtca ctcaagtgaa acagccaaag ttactgaatt
     1501 tcaagccatt gagggaagcg cttaaggatc caggggattt tctatttagt gatttctcta
     1561 agtttgatcg gcctccactc cttcacttag cattccaggc acttgatcat tttaaagctg
     1621 aagctggcag atttcctgtt gctggctcag aagaggacgc tcagaagctt atatcgattg
     1681 ccacagccat caataccggg cagggtgatt taaaggtgga gaatgttgat cagaagcttc
     1741 taagacactt ttcctttgga gccaaggctg tcctgaatcc tatggctgca atgtttggcg
     1801 gtattgttgg acaggaggtt gttaaagctt gctctggaaa atttcatcct ctcttccagt
     1861 ttttctactt tgattcagtg gagtcactcc cctctgaacc tgtggactcg agtgactttg
     1921 caccaagaaa cagccggtat gatgcccaaa tatctgtatt tggggccaag ttccagaaga
     1981 aacttgaaga tgctaaagtt ttcacagtag ggtctggtgc tcttggctgt gagttcttga
     2041 aaaatttggc tctgatgggg gtttcttgtg gaagccaagg gaagctgaca gtgacagatg
     2101 atgatatcat tgagaagagt aacctcagtc gtcaatttct gttccgtgac tggaacattg
     2161 gacaggctaa atccacagtt gctgcttcgg ctgctgctgt tataaatcct cgcttcaaca
     2221 ttgaggccct gcaaaaccgt gttggcgcgg agactgagaa tgtatttgac gatgccttct
     2281 gggagaactt aactgttgtc gtcaatgcac tggataatgt taatgcgagg ctctatgttg
     2341 attcgaggtg cttgtatttc cagaagcctc tccttgagtc tggaactctt ggtacaaagt
     2401 gcaacacaca gtcggttatc ccacatctaa ctgaaaatta cggtgcctca agggacccac
     2461 cagagaaaca ggcccccatg tgtacagtgc attccttccc gcataacatt gatcactgtt
     2521 taacttgggc tcgctctgag tttgagggtc tgctggagaa aactcccgct gaagtgaatg
     2581 catatctttc tagcccagtt gagtacacta actcaatgat gagtgctggt gatgctcagg
     2641 cgagggacac attggagagg attgttgagt gccttgaaaa ggagaagtgt gaaaccttcc
     2701 aagactgctt aacctgggca cgactgaggt ttgaggatta ctttgtgaac cgtgtcaagc
     2761 aattgatata cacatttcct gaagatgctg cgactagtac cggagctcca ttctggtctg
     2821 ctccaaaaag attccctcgt ccactccagt actcgtcgtc cgacccaagt ctccttaact
     2881 ttatcacagc gactgctatt ttacgagcag agacatttgg gatcccgata cctgagtgga
     2941 ctaagaaccc aaaggaagca gcagaagctg ttgacagagt gatagtccca gactttgagc
     3001 caaggcaaga tgcaaagatt gtgactgatg agaaagctac cactttaacc actgcttcgg
     3061 tggacgacgc tgcagtcatc gacgatctca ttgctaagat cgaccagtgt aggcataact
     3121 tgtcgccaga cttcaggatg aaacctattc agttcgaaaa ggatgatgat acaaactatc
     3181 acatggatgt gatagcagga ctagccaaca tgagggcgag aaattacagc atccctgaag
     3241 tggacaagct gaaagcaaag ttcatagcag ggagaatcat cccagccatt gcgacttcaa
     3301 cagccatggc tactgggctg gtctgcctcg agctgtacaa ggtccttgat ggaggacata
     3361 aagtggaagc ttacagaaac acatttgcca acctggcact tccactattc tccatggctg
     3421 aaccacttcc accaaaggtg gtgaagcacc gtgacatggc ttggacagtt tgggacagat
     3481 gggttctcaa gggaaaccca actcttcgtg aggtgttgca gtggctggag gacaaagggc
     3541 ttagcgcata cagcatctct tgtggaagct gtctactgtt caacagtatg ttcacaaggc
     3601 acaaggagag gatggacaag aaggttgtgg atctcgctcg ggatgttgcg aaagtcgagt
     3661 taccgcctta tcgcaaccat cttgatgtag tggtggcttg tgaggatgaa gatgacaatg
     3721 acgtcgatat tcctctcgtc tctatctact tcaggtgagc ttactattaa ctttaagaag
     3781 gagatatacc atgtacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc
     3841 ggactatgca ggattgtatc catatgacgt tccagattac gccactagag ctgcttaccc
     3901 atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg caggattgta
     3961 tccatatgac gttccagatt acgcaatggc ttcgaaaagg atcttgaaag agctcaagga
     4021 tctccagaag gatcctccaa cttcctgcag tgctggccca gttgctgagg acatgtttca
     4081 ttggcaagct acaataatgg gtccatctga tagtccttat tcaggcggtg tctttctcgt
     4141 aaccattcac ttccctccgg attatccttt caaaccacca aaggttgcat tcaggacaaa
     4201 agtgttccac cctaatgtca acagcaatgg aagcatttgc cttgacattt tgaaagaaca
     4261 atggagtcct gcactcacca tatcgaaggt tttgctttcg atatgttcat tgttaacgga
     4321 cccaaaccca gatgatccat tggttccaga gattgctcac atgtacaaaa ccgatagagc
     4381 aaagtatgag tctactgcga gaagctggac tcagaaatat gcaatgggat gagcttttac
     4441 gcttttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4501 gctcccggca tccgcttaca gacaagctgt gaccgggatg agatccggct gctaacaaag
     4561 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     4621 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     4681 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4741 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4801 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4861 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4921 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4981 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5041 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5101 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5221 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     5281 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     5341 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     5401 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     5461 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     5521 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     5581 attcattcgt gattgcgcct gagcgagtcg aaatacgcga tcgctgttaa aaggacaatt
     5641 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     5701 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     5761 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     5821 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     5881 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     5941 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     6001 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     6061 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     6121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6181 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6241 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6301 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6361 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6421 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6481 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6541 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6601 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6661 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6721 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6781 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6841 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6901 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     6961 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     7021 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     7081 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     7141 gcatccgctt acagacaagc tgtgaccgac tccgggagct gcatgtgtca gaggttttca
     7201 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     7261 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     7321 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     7381 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     7441 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     7501 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     7561 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     7621 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     7681 cggaaaccca agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     7741 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     7801 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     7861 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     7921 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     7981 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     8041 atgataaaga acacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     8101 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     8161 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     8221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     8281 cagggtggtt tttcttttca ccagtgacac gggcaacagc tgattgccct tcaccgcctg
     8341 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     8401 tttgatggtg gttaacggcg ggatataaca tgagctgtcc tcggtatcgt cgtatcccac
     8461 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     8521 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     8581 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     8641 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     8701 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     8761 gcccagtcgc gtaccgtcct catgggagaa aataatactg ttgatgggtg tctggtcaga
     8821 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     8881 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     8941 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     9001 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     9061 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     9121 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     9181 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     9241 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     9301 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     9361 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     9421 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     9481 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcc
//
