LOCUS       sequence-317671-        5203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PKA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5203)
  AUTHORS   Wallace AK, Chanut N, Voigt CA
  TITLE     Silica Nanostructures Produced Using Diatom Peptides with Designed 
            Post-Translational Modifications
  JOURNAL   Advanced Functional Materials 2020
REFERENCE   2  (bases 1 to 5203)
  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"; journalName: "Advanced 
            Functional Materials 2020"
COMMENT     Sequence Label: enz.099-PKA
FEATURES             Location/Qualifiers
     source          1..5203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        34..83
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             130..147
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             160..1257
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNQPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1114..1123
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1231..1254
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1291..1311
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      2387..2458
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2474..2501
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2521..2578
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             complement(2736..3551)
                     /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(2736..3551)
                     /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     3459..3478
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3543..3562
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      complement(4146..4691)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4226..4245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatacgact cactataggg agatactaga gagctgtcaa aggatgtgct ttccggtctg
       61 atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaagaaa gaggggacaa
      121 actagtatgc atcaccatca ccatcacggc ggtagtggca aaatcgaaga aggtaaactg
      181 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
      241 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
      301 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
      361 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
      421 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
      481 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa ccagccaaaa
      541 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
      601 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
      661 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
      721 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
      781 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
      841 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
      901 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
      961 gccgccagtc cgaacaaaga gctggcgaaa gagttcctcg aaaactatct gctgactgat
     1021 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     1081 gaggaagagt tggcgaaaga tccacgtatt gccgccacca tggaaaacgc ccagaaaggt
     1141 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     1201 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactcgt
     1261 atcaccaagg aactggttgc aagtgctaaa gaaaacctgt attttcaggg catgggcaac
     1321 gccgccgccg ccaagaaggg cagcgagcag gagagcgtga aagagttcct agccaaagcc
     1381 aaggaagatt tcctgaaaaa atgggaaacc ccttctcaga atacagccca gttggatcag
     1441 tttgatagaa tcaagaccct tggcaccggc tcctttgggc gagtgatgct ggtgaagcac
     1501 aaggagagtg ggaaccacta cgccatgaag atcttagaca agcagaaggt ggtgaagcta
     1561 aagcagatcg agcacactct gaatgagaag cgcatcctgc aggccgtcaa cttcccgttc
     1621 ctggtcaaac ttgaattctc cttcaaggac aactcaaacc tgtacatggt catggagtat
     1681 gtagctggtg gcgagatgtt ctcccaccta cggcggattg gaaggttcag cgagccccat
     1741 gcccgtttct acgcggcgca gatcgtcctg acctttgagt atctgcactc cctggacctc
     1801 atctaccggg acctgaagcc cgagaatctt ctcatcgacc agcagggcta tattcaggtg
     1861 acagacttcg gttttgccaa gcgtgtgaaa ggccgtactt ggaccttgtg tgggacccct
     1921 gagtacttgg cccccgagat tatcctgagc aaaggctaca acaaggctgt ggactggtgg
     1981 gctctcggag tcctcatcta cgagatggct gctggttacc cacccttctt cgctgaccag
     2041 cctatccaga tctatgagaa aatcgtctct gggaaggtgc ggttcccatc ccacttcagc
     2101 tctgacttga aggacctgct gcggaacctt ctgcaagtgg atctcaccaa gcgctttggg
     2161 aacctcaaga acggggtcaa tgacatcaag aaccacaagt ggtttgccac gactgactgg
     2221 attgccatct atcagagaaa ggtggaagct cccttcatac caaagtttaa aggccctggg
     2281 gacacgagta actttgacga ctatgaggag gaagagatcc gggtttccat caatgagaag
     2341 tgtggcaagg agtttactga gttttaataa tactagagcc aggcatcaaa taaaacgaaa
     2401 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     2461 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatatac tagtagccag
     2521 tccggcaaaa aagggcaagg tgtcaccacc ctgccctttt tctttaaaac cgaaaagatt
     2581 acttcgcgtt atgcaggctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2641 gcgagcggta tcagctcact caaaggcggt aatctcgagt cccgtcaagt cagcgtaatg
     2701 ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     2761 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2821 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2881 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2941 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     3001 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     3061 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     3121 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3181 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3241 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3301 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3361 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3421 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3481 tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga
     3541 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3601 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     3661 tgttgaataa atcgaacttt tgctgagttg aaggatcaga tcacgcatct tcccgacaac
     3721 gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca actggtccac ctacaacaaa
     3781 gctctcatca accgtggctc cctcactttc tggctggatg atggggcgat tcaggcctgg
     3841 tatgagtcag caacaccttc ttcacgaggc agacctcagc gctagcggag tgtatactgg
     3901 cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa
     3961 ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac
     4021 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
     4081 agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag
     4141 ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag
     4201 tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc
     4261 gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt
     4321 ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg
     4381 tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga
     4441 gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta attgatttag
     4501 aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg
     4561 actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg
     4621 aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa
     4681 aacgatctca agaagatcat cttattaagg ggtctgacgc tcagtggaac gaaaactcac
     4741 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4801 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4861 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4921 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4981 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5041 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5101 ttccatggtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     5161 taggtgtatc acgagtcgct tgaattcgct gcagcttcta gag
//
