LOCUS       sequence-404767-        5119 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For recombinant protein production in Escherichia coli. His-tagged 
            UnaG under the control of the T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5119)
  AUTHORS   Ishikawa K, Xie X, Osaki Y, Miyawaki A, Numata K, Kodama Y
  TITLE     Bilirubin is produced nonenzymatically in plants to maintain 
            chloroplast redox status.
  JOURNAL   Sci Adv. 2023 Jun 9;9(23):eadh4787. doi: 10.1126/sciadv.adh4787. 
            Epub 2023 Jun 7.
  PUBMED    37285441
REFERENCE   2  (bases 1 to 5119)
  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: "Sci Adv."; 
            date: "2023-06-9"; volume: "9(23):eadh4787. doi"; pages: " 
            10.1126/sciadv.adh4787. Epub 2023 Jun 7"
COMMENT     Sequence Label: pDEST17-UnaG
FEATURES             Location/Qualifiers
     source          1..5119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             71..93
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             113..130
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    140..164
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             167..586
                     /codon_start=1
                     /product="bilirubin-binding green fluorescent protein from 
                     Anguilla japonica (Kumagai et al., 2013)"
                     /label=UnaG
                     /translation="MVEKFVGTWKIADSHNFGEYLKAIGAPKELSDGGDATTPTLYISQ
                     KDGDKMTVKIENGPPTFLDTQVKFKLGEEFDEFPSDRRKGVKSVVNLVGEKLVYVQKWD
                     GKETTYVREIKDGKLVVTLTMGDVVAVRSYRRATE"
     protein_bind    complement(588..612)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(665..683)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      679..726
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1092..1097,1098..1114,1115..1120))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1147..1165)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1233..1337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1338..1406,1407..2198)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1556..1575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2369..2957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2858..2877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3111..3128
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3143..3282
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3208..3227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3368..3390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3384..3575)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5037..5056
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
       61 tagaaataat tttgtttaac tttaagaagg agatatacat atgtcgtact accatcacca
      121 tcaccatcac ctcgaatcaa caagtttgta caaaaaagca ggcttcatgg tcgagaaatt
      181 tgttggcacc tggaagatcg cagacagcca taattttggt gaatacctga aagctatcgg
      241 agccccaaag gaattaagcg atggtgggga tgccacgacg ccgacattgt acatctccca
      301 gaaggacgga gacaaaatga cagtgaaaat agagaatgga cctcctacgt tccttgacac
      361 tcaagtaaag ttcaaattag gggaggagtt cgacgaattt ccttctgatc gaagaaaagg
      421 cgtaaaatct gtcgtgaact tggtgggaga gaagctggtg tacgtacaaa agtgggacgg
      481 caaggagacg acgtatgtcc gagagataaa ggacggtaaa ctggtcgtga cacttacgat
      541 gggagacgtc gtggctgtgc gcagctaccg gagggcgacg gaatgagacc cagctttctt
      601 gtacaaagtg gttgattcga ggctgctaac aaagcccgaa aggaagctga gttggctgct
      661 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      721 tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat
      781 cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg
      841 gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag
      901 cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg
      961 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     1021 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     1081 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     1141 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1201 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1261 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1321 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1381 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1441 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1501 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1561 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1621 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1681 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1741 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1801 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1861 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1921 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     1981 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2041 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2101 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2161 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2221 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2281 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2341 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2401 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2461 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2521 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2581 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2641 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2701 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2761 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2821 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2881 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     2941 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3001 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3061 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3121 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3181 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3241 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3301 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3361 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3421 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3481 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3541 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3601 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3661 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3721 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3781 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3841 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     3901 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     3961 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4021 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     4081 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     4141 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     4201 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     4261 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     4321 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     4381 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     4441 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     4501 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     4561 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4621 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4681 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4741 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4801 agctgactgg gttgaaggct ctcaagggca tcggtcgatc gacgctctcc cttatgcgac
     4861 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     4921 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     4981 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5041 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5101 cgtccggcgt agaggatcg
//
