LOCUS       Exported                9529 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Level 2 construct.
ACCESSION   .
VERSION     .
KEYWORDS    pUG-352
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9529)
  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 9529)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9529
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(560..1375)
                     /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     1283..1302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1367..1386
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1986..2005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2239..2256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2339..2358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2499..2521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /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     4559..4578
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4881..4900
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4984..5003
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /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             5081..5098
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             8646..8732
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             8709..8735
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8748..8834
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY"
     CDS             8811..8837
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(9443..9461)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      9457..9504
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gtcgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgactcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaacccaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaca cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgacacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcctcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcctcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgcctta actttaagaa ggagatatac catgggcagc caccaccacc accaccacag
     5101 cggaatgcag atctttgtta agactctcac cggaaagaca atcaccctgg aggtggaaag
     5161 ctccgacacc atcgacaacg ttaaggccaa gatccaggat aaggagggca ttcctccgga
     5221 tcagcagagg cttattttcg ccggcaagca gctagaggat ggccgtacgt tggctgatta
     5281 caatatccag aaggaatcca ccctccactt ggtcctcagg ctccgtggtg gttaagcttg
     5341 caattaactt taagaaggag atatacaatg cttcacaagc gagctagtga agctaacgat
     5401 aagaacgata ataccatcat cggttccgat ttagcatcat ctaagaaacg ccgtatcgat
     5461 ttcactgaat cttcgtctga taagtcatct tcgattttag cttctggtag tagcaggggt
     5521 tttcacggcg atagcgtcgt tcagcaaatc gacatggctt ttggtaattc gaaccgtcag
     5581 gagattgatg aagatctgca cagtcgccag ctcgccgtct atggtcgtga gactatgagg
     5641 cgtctctttg cttccaatgt tctcatctcg gggatgcacg gtcttggtgc cgagattgcc
     5701 aagaatctta tacttgctgg tgtgaagtct gtgaccttgc atgatgaaag agttgtagag
     5761 ctatgggact tatcaagcaa ctttgttttc tcggaggatg atgttggcaa gaatagagca
     5821 gatgcttctg ttcagaagtt acaggatctt aacaatgctg tggttgtctc tagcttgacc
     5881 aaaagcttaa acaaagagga tctttctggc ttccaggttg ttgtattctc tgacataagc
     5941 atggaaagag caatcgaatt tgatgactat tgtcacagcc atcagcctcc tatcgctttt
     6001 gtcaaggctg atgtcagggg tcttttcggc tctgtctttt gtgattttgg gcctgaattt
     6061 gcggttcttg atgttgatgg agaggaaccc catacaggca tcattgcgtc tatctctaat
     6121 gagaaccagg catttatttc ctgtgtcgac gatgagaggc ttgagtttga agatggtgac
     6181 ctagttgttt tctcagaagt tgagggtatg acagaattga atgatgggaa accaaggaag
     6241 ataaaaagta cacggccata ttcattcacc cttgatgagg acactactaa ctatggaaca
     6301 tatgtgaagg gtggtattgt cactcaagtg aaacagccaa agttactgaa tttcaagcca
     6361 ttgagggaag cgcttaagga tccaggggat tttctattta gtgatttctc taagtttgat
     6421 cggcctccac tccttcactt agcattccag gcacttgatc attttaaagc tgaagctggc
     6481 agatttcctg ttgctggctc agaagaggac gctcagaagc ttatatcgat tgccacagcc
     6541 atcaataccg ggcagggtga tttaaaggtg gagaatgttg atcagaagct tctaagacac
     6601 ttttcctttg gagccaaggc tgtcctgaat cctatggctg caatgtttgg cggtattgtt
     6661 ggacaggagg ttgttaaagc ttgctctgga aaatttcatc ctctcttcca gtttttctac
     6721 tttgattcag tggagtcact cccctctgaa cctgtggact cgagtgactt tgcaccaaga
     6781 aacagccggt atgatgccca aatatctgta tttggggcca agttccagaa gaaacttgaa
     6841 gatgctaaag ttttcacagt agggtctggt gctcttggct gtgagttctt gaaaaatttg
     6901 gctctgatgg gggtttcttg tggaagccaa gggaagctga cagtgacaga tgatgatatc
     6961 attgagaaga gtaacctcag tcgtcaattt ctgttccgtg actggaacat tggacaggct
     7021 aaatccacag ttgctgcttc ggctgctgct gttataaatc ctcgcttcaa cattgaggcc
     7081 ctgcaaaacc gtgttggcgc ggagactgag aatgtatttg acgatgcctt ctgggagaac
     7141 ttaactgttg tcgtcaatgc actggataat gttaatgcga ggctctatgt tgattcgagg
     7201 tgcttgtatt tccagaagcc tctccttgag tctggaactc ttggtacaaa gtgcaacaca
     7261 cagtcggtta tcccacatct aactgaaaat tacggtgcct caagggaccc accagagaaa
     7321 caggccccca tgtgtacagt gcattccttc ccgcataaca ttgatcactg tttaacttgg
     7381 gctcgctctg agtttgaggg tctgctggag aaaactcccg ctgaagtgaa tgcatatctt
     7441 tctagcccag ttgagtacac taactcaatg atgagtgctg gtgatgctca ggcgagggac
     7501 acattggaga ggattgttga gtgccttgaa aaggagaagt gtgaaacctt ccaagactgc
     7561 ttaacctggg cacgactgag gtttgaggat tactttgtga accgtgtcaa gcaattgata
     7621 tacacatttc ctgaagatgc tgcgactagt accggagctc cattctggtc tgctccaaaa
     7681 agattccctc gtccactcca gtactcgtcg tccgacccaa gtctccttaa ctttatcaca
     7741 gcgactgcta ttttacgagc agagacattt gggatcccga tacctgagtg gactaagaac
     7801 ccaaaggaag cagcagaagc tgttgacaga gtgatagtcc cagactttga gccaaggcaa
     7861 gatgcaaaga ttgtgactga tgagaaagct accactttaa ccactgcttc ggtggacgac
     7921 gctgcagtca tcgacgatct cattgctaag atcgaccagt gtaggcataa cttgtcgcca
     7981 gacttcagga tgaaacctat tcagttcgaa aaggatgatg atacaaacta tcacatggat
     8041 gtgatagcag gactagccaa catgagggcg agaaattaca gcatccctga agtggacaag
     8101 ctgaaagcaa agttcatagc agggagaatc atcccagcca ttgcgacttc aacagccatg
     8161 gctactgggc tggtctgcct cgagctgtac aaggtccttg atggaggaca taaagtggaa
     8221 gcttacagaa acacatttgc caacctggca cttccactat tctccatggc tgaaccactt
     8281 ccaccaaagg tggtgaagca ccgtgacatg gcttggacag tttgggacag atgggttctc
     8341 aagggaaacc caactcttcg tgaggtgttg cagtggctgg aggacaaagg gcttagcgca
     8401 tacagcatct cttgtggaag ctgtctactg ttcaacagta tgttcacaag gcacaaggag
     8461 aggatggaca agaaggttgt ggatctcgct cgggatgttg cgaaagtcga gttaccgcct
     8521 tatcgcaacc atcttgatgt agtggtggct tgtgaggatg aagatgacaa tgacgtcgat
     8581 attcctctcg tctctatcta cttcaggtga gcttactatt aactttaaga aggagatata
     8641 ccatgtaccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg
     8701 caggattgta tccatatgac gttccagatt acgccactag agctgcttac ccatacgatg
     8761 ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggattg tatccatatg
     8821 acgttccaga ttacgcaatg gcttcgaaac ggatcttgaa ggagctgaag gatctccaga
     8881 aagaccctcc aacctcctgc agtgcaggtc cagttgctga ggacatgttt cattggcaag
     8941 ctacaattat gggtcctgca gagagtccgt attcaggcgg tgtgtttctc gttaccattc
     9001 acttccctcc ggactatcca ttcaaaccac caaaggttgc atttaggacg aaggtgtttc
     9061 accctaatat caacagcaac ggaagcattt gccttgacat tttgaaagaa caatggagcc
     9121 ctgccctcac catttccaag gtgttgctct cgatatgttc gctgttaaca gatccaaatc
     9181 cagatgaccc tttggtacca gagattgcac acatgtacaa aaccgacaga gccaaatacg
     9241 aggctactgc aagaaactgg actcagaagt atgccatggg ctaagctttt acgcttttaa
     9301 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     9361 catccgctta cagacaagct gtgaccggga tgagatccgg ctgctaacaa agcccgaaag
     9421 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     9481 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat
//
