LOCUS       sequence-205469-        5779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 2 cloning vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5779)
  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 5779)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1111/nph.14900";
            journalName: "New Phytol"; date: "2018-03"; volume: "217"; issue: 
            "4"; pages: "1749-1763"
COMMENT     Sequence Label: pET-28GG
FEATURES             Location/Qualifiers
     source          1..5779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..25
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     109..128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        109..127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    128..152
                     /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)."
     protein_bind    236..257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(272..277,278..295,296..302)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    310..326
                     /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)."
     primer_bind     315..337
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     334..350
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     334..350
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             346..669
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    363..419
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(420..437)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(420..436)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(429..451)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(822..840)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      836..883
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      920..1375
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1007..1026)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1217..1238
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1467..2282)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1467..2282)
                     /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     2190..2209
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2274..2293
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2404..2992
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2893..2912
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3146..3163
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3178..3317
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3403..3425
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3419..3610)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4419..5501)
                     /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     5463..5482
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5502..5579)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
       61 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
      121 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg ccttgtcttc
      181 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      241 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      301 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      361 ccaagcttgc atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattca
      421 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      481 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
      541 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
      601 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
      661 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
      721 tgtctgctcc cggcatccgc ttacagacaa gctgtgagaa gacaagggat gagatccggc
      781 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
      841 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
      901 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
      961 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1021 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1081 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1141 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1201 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1261 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1321 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     1381 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1441 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     1501 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1561 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1621 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1681 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1741 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1801 accaaaccgt tattcattcg tgattgcgcc tgagcgagtc gaaatacgcg atcgctgtta
     1861 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1921 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1981 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2041 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2101 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2161 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2221 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2281 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     2341 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2401 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2461 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2521 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     2581 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2641 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2701 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2761 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2821 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2881 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2941 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3001 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3061 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3121 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3181 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     3241 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3301 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3361 gtctgctccc ggcatccgct tacagacaag ctgtgaccga ctccgggagc tgcatgtgtc
     3421 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3481 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3541 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3601 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3661 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3721 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     3781 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     3841 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     3901 tttacgaaac acggaaaccc aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     3961 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4021 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4081 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4141 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4201 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4261 ctacgagttg catgataaag aacacagtca taagtgcggc gacgatagtc atgccccgcg
     4321 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4381 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4441 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4501 gtattgggcg ccagggtggt ttttcttttc accagtgaca cgggcaacag ctgattgccc
     4561 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4621 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ctcggtatcg
     4681 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4741 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4801 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4861 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4921 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4981 agatgctcca cgcccagtcg cgtaccgtcc tcatgggaga aaataatact gttgatgggt
     5041 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5101 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5161 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5221 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5281 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5341 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5401 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5461 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5521 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5581 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5641 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5701 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5761 gtggcgagcc cgatcttcc
//
