LOCUS       sequence-350010-        6715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of SnRK2.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  AUTHORS   Ng LM, Soon FF, Zhou XE, West GM, Kovach A, Suino-Powell KM, 
            Chalmers MJ, Li J, Yong EL, Zhu JK, Griffin PR, Melcher K, Xu HE
  TITLE     Structural basis for basal activity and autoactivation of abscisic 
            acid (ABA) signaling SnRK2 kinases.
  JOURNAL   Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21259-64. doi: 
            10.1073/pnas.1118651109. Epub 2011 Dec 12.
  PUBMED    22160701
REFERENCE   2  (bases 1 to 6715)
  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: 
            "10.1073/pnas.1118651109"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2011-12-27- 27"; volume: "108"; issue: "52"; pages: 
            "21259-64"
COMMENT     Sequence Label: pSUMO His6 SUMO SnRK2.2 (20-362)
FEATURES             Location/Qualifiers
     source          1..6715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1293..1310
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1363..1381)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1377..1424
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1461..1916
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1548..1567)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1758..1779
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1942..2046
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2047..2115,2116..2907)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2265..2284)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3078..3666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3567..3586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3820..3837
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3852..3991
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3917..3936)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4077..4099
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4093..4284)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5093..6175)
                     /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     6137..6156
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6176..6253)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6459..6478
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6562..6581
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6562..6580
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6581..6605
                     /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)."
     RBS             6620..6642
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6655..6672
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(6676..6715,1..251)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
       61 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
      121 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
      181 atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac
      241 agattggagg tggaagtgat cgttacgact tcgttaaaga tattggctct ggtaatttcg
      301 gcgttgctcg tctcatgacc gatagagtca ccaaggagct tgttgctgtt aaatacatcg
      361 agagaggaga aaagattgat gaaaatgttc agagggagat tatcaatcat agatcattga
      421 gacatcctaa tattgttagg tttaaagagg tgattttgac gccttcccat ttggctattg
      481 ttatggaata tgctgctggt ggagaacttt atgagcggat ttgtaatgcc ggacggttta
      541 gtgaagatga ggctcggttc ttctttcagc agcttatatc tggagttagc tattgtcatg
      601 caatgcaaat atgccatcgg gatctgaagc tggaaaatac attgttagat ggaagtccgg
      661 cacctcgttt gaaaatatgt gattttggtt attccaagtc ttctgttctt cattcccaac
      721 caaagtcaac tgttggtact cctgcataca ttgcaccaga gattcttctt cgacaggaat
      781 atgatggcaa gcttgcagat gtatggtctt gcggtgtaac attatatgta atgttggttg
      841 gagcttatcc attcgaggat ccacaggagc cacgagatta tcgaaagaca atacaaagaa
      901 tccttagtgt cacatactcg atcccagagg acttacacct ctcaccagaa tgtcgccatc
      961 taatatcgag gatcttcgtg gctgatccgg caacaagaat cactattccg gagatcacat
     1021 ccgataaatg gttcttgaag aatctaccag gtgatttgat ggatgagaac cgaatgggaa
     1081 gtcagtttca agagcctgag cagccaatgc agagccttga cacgattatg cagataatat
     1141 cggaggctac gattccgact gttcgtaatc gttgcctcga tgatttcatg gcggataatc
     1201 ttgatctaga cgatgacatg gatgactttg attccgaatc tgagattgat gttgacagta
     1261 gtggagagat agtttatgct ctctgactcg agcaccacca ccaccaccac tgagatccgg
     1321 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1381 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1441 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1501 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1561 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1621 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1681 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1741 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1801 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1861 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     1921 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1981 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2041 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2101 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2161 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2221 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2281 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2341 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2401 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2461 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2521 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2581 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2641 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2701 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2761 ggttctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2821 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2881 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2941 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3001 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3061 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3121 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3181 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3241 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3301 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3361 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3421 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3481 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3541 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3601 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3661 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3721 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3781 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3841 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3901 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3961 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4021 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4081 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4141 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4201 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4261 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4321 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4381 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4441 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4501 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4561 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4621 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4681 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4741 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4801 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4861 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4921 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4981 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5041 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5101 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5161 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     5221 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     5281 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5341 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     5401 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5461 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5521 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5581 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5641 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5701 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5761 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5821 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5881 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5941 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     6001 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6061 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6121 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     6181 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     6241 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6301 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6361 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6421 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6481 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6541 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     6601 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggtcatcac
     6661 catcatcatc acgggtcgga ctcagaagtc aatcaagaag ctaagccaga ggtca
//
