LOCUS       Exported                9564 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Level 2 construct.
ACCESSION   .
VERSION     .
KEYWORDS    pUG-306
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     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1254..1273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1376..1400
                     /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             1454..1471
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5019..5105
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             5082..5108
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5121..5207
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             5184..5210
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5855..5873)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5869..5916
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5953..6408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6040..6059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6250..6271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6500..7315)
                     /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     7223..7242
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7307..7326
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7437..8025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7926..7945
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8179..8196
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8211..8350
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8276..8295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8436..8458
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8452..8643)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(9452..970)
                     /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 ccagtgacac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtcc tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtcct
      481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1381 tgtgagcgga taacaattcc cctctagaaa taattttgcc ttaactttaa gaaggagata
     1441 taccatgggc agccaccacc accaccacca cagcggaatg cagatctttg ttaagactct
     1501 caccggaaag acaatcaccc tagaggtgga aagctccgac accatcgaca acgttaaggc
     1561 caagatccag gataaggagg gcattcctcc ggatcagcag aggcttattt tcgccggcaa
     1621 gcagctagag gatggccgta cgttggctga ttacaatatc cagaaggaat ccaccctcca
     1681 cttggtcctc aggctccgtg gtggttaagc ttgcaattaa ctttaagaag gagatataca
     1741 atgcttcaca agcgagctag tgaagctaac gataagaacg ataataccat catcggttcc
     1801 gatttagcat catctaagaa acgccgtatc gatttcactg aatcttcgtc tgataagtca
     1861 tcttcgattt tagcttctgg tagtagcagg ggttttcacg gcgatagcgt cgttcagcaa
     1921 atcgacatgg cttttggtaa ttcgaaccgt caggagattg atgaagatct gcacagtcgc
     1981 cagctcgccg tctatggtcg tgagactatg aggcgtctct ttgcttccaa tgttctcatc
     2041 tcggggatgc acggtcttgg tgccgagatt gccaagaatc ttatacttgc tggtgtgaag
     2101 tctgtgacct tgcatgatga aagagttgta gagctatggg acttatcaag caactttgtt
     2161 ttctcggagg atgatgttgg caagaataga gcagatgctt ctgttcagaa gttacaggat
     2221 cttaacaatg ctgtggttgt ctctagcttg accaaaagct taaacaaaga ggatctttct
     2281 ggcttccagg ttgttgtatt ctctgacata agcatggaaa gagcaatcga atttgatgac
     2341 tattgtcaca gccatcagcc tcctatcgct tttgtcaagg ctgatgtcag gggtcttttc
     2401 ggctctgtct tttgtgattt tgggcctgaa tttgcggttc ttgatgttga tggagaggaa
     2461 ccccatacag gcatcattgc gtctatctct aatgagaacc aggcatttat ttcctgtgtc
     2521 gacgatgaga ggcttgagtt tgaagatggt gacctagttg ttttctcaga agttgagggt
     2581 atgacagaat tgaatgatgg gaaaccaagg aagataaaaa gtacacggcc atattcattc
     2641 acccttgatg aggacactac taactatgga acatatgtga agggtggtat tgtcactcaa
     2701 gtgaaacagc caaagttact gaatttcaag ccattgaggg aagcgcttaa ggatccaggg
     2761 gattttctat ttagtgattt ctctaagttt gatcggcctc cactccttca cttagcattc
     2821 caggcacttg atcattttaa agctgaagct ggcagatttc ctgttgctgg ctcagaagag
     2881 gacgctcaga agcttatatc gattgccaca gccatcaata ccgggcaggg tgatttaaag
     2941 gtggagaatg ttgatcagaa gcttctaaga cacttttcct ttggagccaa ggctgtcctg
     3001 aatcctatgg ctgcaatgtt tggcggtatt gttggacagg aggttgttaa agcttgctct
     3061 ggaaaatttc atcctctctt ccagtttttc tactttgatt cagtggagtc actcccctct
     3121 gaacctgtgg actcgagtga ctttgcacca agaaacagcc ggtatgatgc ccaaatatct
     3181 gtatttgggg ccaagttcca gaagaaactt gaagatgcta aagttttcac agtagggtct
     3241 ggtgctcttg gctgtgagtt cttgaaaaat ttggctctga tgggggtttc ttgtggaagc
     3301 caagggaagc tgacagtgac agatgatgat atcattgaga agagtaacct cagtcgtcaa
     3361 tttctgttcc gtgactggaa cattggacag gctaaatcca cagttgctgc ttcggctgct
     3421 gctgttataa atcctcgctt caacattgag gccctgcaaa accgtgttgg cgcggagact
     3481 gagaatgtat ttgacgatgc cttctgggag aacttaactg ttgtcgtcaa tgcactggat
     3541 aatgttaatg cgaggctcta tgttgattcg aggtgcttgt atttccagaa gcctctcctt
     3601 gagtctggaa ctcttggtac aaagtgcaac acacagtcgg ttatcccaca tctaactgaa
     3661 aattacggtg cctcaaggga cccaccagag aaacaggccc ccatgtgtac agtgcattcc
     3721 ttcccgcata acattgatca ctgtttaact tgggctcgct ctgagtttga gggtctgctg
     3781 gagaaaactc ccgctgaagt gaatgcatat ctttctagcc cagttgagta cactaactca
     3841 atgatgagtg ctggtgatgc tcaggcgagg gacacattgg agaggattgt tgagtgcctt
     3901 gaaaaggaga agtgtgaaac cttccaagac tgcttaacct gggcacgact gaggtttgag
     3961 gattactttg tgaaccgtgt caagcaattg atatacacat ttcctgaaga tgctgcgact
     4021 agtaccggag ctccattctg gtctgctcca aaaagattcc ctcgtccact ccagtactcg
     4081 tcgtccgacc caagtctcct taactttatc acagcgactg ctattttacg agcagagaca
     4141 tttgggatcc cgatacctga gtggactaag aacccaaagg aagcagcaga agctgttgac
     4201 agagtgatag tcccagactt tgagccaagg caagatgcaa agattgtgac tgatgagaaa
     4261 gctaccactt taaccactgc ttcggtggac gacgctgcag tcatcgacga tctcattgct
     4321 aagatcgacc agtgtaggca taacttgtcg ccagacttca ggatgaaacc tattcagttc
     4381 gaaaaggatg atgatacaaa ctatcacatg gatgtgatag caggactagc caacatgagg
     4441 gcgagaaatt acagcatccc tgaagtggac aagctgaaag caaagttcat agcagggaga
     4501 atcatcccag ccattgcgac ttcaacagcc atggctactg ggctggtctg cctcgagctg
     4561 tacaaggtcc ttgatggagg acataaagtg gaagcttaca gaaacacatt tgccaacctg
     4621 gcacttccac tattctccat ggctgaacca cttccaccaa aggtggtgaa gcaccgtgac
     4681 atggcttgga cagtttggga cagatgggtt ctcaagggaa acccaactct tcgtgaggtg
     4741 ttgcagtggc tggaggacaa agggcttagc gcatacagca tctcttgtgg aagctgtcta
     4801 ctgttcaaca gtatgttcac aaggcacaag gagaggatgg acaagaaggt tgtggatctc
     4861 gctcgggatg ttgcgaaagt cgagttaccg ccttatcgca accatcttga tgtagtggtg
     4921 gcttgtgagg atgaagatga caatgacgtc gatattcctc tcgtctctat ctacttcagg
     4981 tgagcttact attaacttta agaaggagat ataccatgta cccatacgat gttcctgact
     5041 atgcgggcta tccctatgac gtcccggact atgcaggatt gtatccatat gacgttccag
     5101 attacgccac tagagctgct tacccatacg atgttcctga ctatgcgggc tatccctatg
     5161 acgtcccgga ctatgcagga ttgtatccat atgacgttcc agattacgca atgaccagct
     5221 cttccgctcc ttcacgcaag gctttaagca agatcgcgtg taataggttg cagaaagagc
     5281 tttctgagtg gcaattgaat ccccccactg gatttcgtca caaagtcact gataatcttc
     5341 aaaaatggac aatagatgtt accggagctc cagggacact ttacgctaat gagacttatc
     5401 agcttcaggt tgaatttcct gaacattacc ctatggaagc accccaggta gtgtttgttt
     5461 ctccggcacc ttcacatcca catatataca gcaatggaca tatttgttta gatattctat
     5521 atgactcatg gtcaccagca atgacagtga attcagtctg catcagcatt ctttcgatgc
     5581 tatcaagttc acccgcaaag caacgccctg cggataacga tcgttatgtg aagaactgta
     5641 agaatgggag gtcacctaag gagacgaggt ggtggttcca tgacgacaag gtttgagctt
     5701 ttacgctttt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5761 gtctgctccc ggcatccgct tacagacaag ctgtgaccgg gatgagatcc ggctgctaac
     5821 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     5881 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     5941 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6001 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     6061 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     6121 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     6181 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     6241 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     6301 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6361 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     6421 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6481 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     6541 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     6601 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     6661 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     6721 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     6781 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     6841 cgttattcat tcgtgattgc gcctgagcga gtcgaaatac gcgatcgctg ttaaaaggac
     6901 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     6961 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     7021 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     7081 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     7141 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     7201 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     7261 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     7321 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     7381 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7441 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7501 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7561 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     7621 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7681 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7741 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7801 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7861 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7921 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7981 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     8041 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     8101 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     8161 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     8221 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     8281 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     8341 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     8401 cccggcatcc gcttacagac aagctgtgac cgactccggg agctgcatgt gtcagaggtt
     8461 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     8521 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     8581 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     8641 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     8701 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     8761 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     8821 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     8881 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     8941 aacacggaaa cccaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     9001 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     9061 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     9121 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     9181 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     9241 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     9301 ttgcatgata aagaacacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     9361 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     9421 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     9481 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     9541 gcgccagggt ggtttttctt ttca
//
