LOCUS       sequence-319759-        3844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Arabidopsis thaliana VERNALIZATION1 mutant, with R249E, 
            R289E and R296E mutations, in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3844)
  AUTHORS   King GJ, Chanson AH, McCallum EJ, Ohme-Takagi M, Byriel K, Hill JM, 
            Martin JL, Mylne JS
  TITLE     The Arabidopsis B3 domain protein VERNALIZATION1 (VRN1) is involved 
            in processes essential for development, with structural and 
            mutational studies revealing its DNA-binding surface.
  JOURNAL   J Biol Chem. 2013 Feb 1;288(5):3198-207. doi: 
            10.1074/jbc.M112.438572. Epub 2012 Dec 19.
  PUBMED    23255593
REFERENCE   2  (bases 1 to 3844)
  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: 
            "10.1074/jbc.M112.438572"; journalName: "J Biol Chem"; date: 
            "2013-02-1- 1"; volume: "288"; issue: "5"; pages: "3198-207"
COMMENT     Sequence Label: pQE30-VRN1-R249E-R289E-R296E
FEATURES             Location/Qualifiers
     source          1..3844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..15,16..21,22..38,39..45)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    21..37
                     /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    53..69
                     /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             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      582..676
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             720..1379
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(786..805)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1444..1530
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1577..1599)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1685..1825
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1740..1759
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1840..1857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2011..2599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2091..2110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2770..3561,3562..3630))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3393..3412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3631..3735)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3803..3821
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagattc aattgtgagc
       61 ggataacaat ttcacacaga attcattaaa gaggagaaat taactatgag aggatcgcat
      121 caccatcacc atcacggatc ccgttcaaag ttctacgaga gtgcttctgc gagaaagaga
      181 accgtgactg cagaagaaag agagagagcc atcaatgcag ccaaaacgtt cgaaccaaca
      241 aaccctttct tcagagtggt tctggagcca tcctatctat acagaggttg catcatgtat
      301 cttccttctg ggtttgctga gaagtaccta agtgggatct ccgggttcat caaagtccag
      361 cttgcggaga aacaatggcc tgttgagtgt ctctacaaag ccggggaggc caaattcagt
      421 caaggatggt acgaattcac tctagagaac aacttaggag aaggagacgt ctgtgtgttt
      481 gagctgctca gaaccagaga tttcgttttg aaagtgacag cctttcgagt caacgagtac
      541 gtctgagtcg acctgcagcc aagcttaatt agctgagctt ggactcctgt tgatagatcc
      601 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
      661 ttttattggt gagaatccaa gctagcttgg cgagattttc aggagctaag gaagctaaaa
      721 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
      781 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
      841 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
      901 acattcttgc ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg
      961 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     1021 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     1081 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     1141 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     1201 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     1261 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     1321 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat
     1381 ttttttaagg cagttattgg tgcccttaaa cgcctggggt aatgactctc tagcttgagg
     1441 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     1501 tcggtgaacg ctctcctgag taggacaaat ccgccctcta gagctgcctc gcgcgtttcg
     1561 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     1621 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     1681 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     1741 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     1801 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     1861 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1921 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     1981 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2041 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2101 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2161 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2221 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2281 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2341 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2401 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     2461 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2521 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2581 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2641 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2701 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2761 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2821 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2881 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     2941 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3001 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3061 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3601 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     3661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     3781 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcacctcga
     3841 gaaa
//
