LOCUS       sequence-417678-        5951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the bright and photostable monomeric 
            StayGold fluorescent protein (E138D). Codon optimised for expression
            in Escherichia coli. Contains a N-terminal 10xHis tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5951)
  AUTHORS   Ivorra-Molla E, Akhuli D, McAndrew MBL, Scott W, Kumar L, Palani S, 
            Mishima M, Crow A, Balasubramanian MK
  TITLE     A monomeric StayGold fluorescent protein.
  JOURNAL   Nat Biotechnol. 2023 Dec 11. doi: 10.1038/s41587-023-02018-w.
  PUBMED    38081970
REFERENCE   2  (bases 1 to 5951)
  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: "Nat 
            Biotechnol. 2023 Dec 11. doi: 10.1038/s41587-023-02018-w."
COMMENT     Sequence Label: pET-MCN-10His-mStayGold (E138D)
FEATURES             Location/Qualifiers
     source          1..5951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(50..638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(130..149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(749..768)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             760..1575
                     /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             760..1575
                     /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     complement(833..852)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1667..2122)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1804..1825)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2016..2035
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2159..2206)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2202..2220
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2322..2972)
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             complement(2322..2972)
                     /codon_start=1
                     /product="bright and highly photostable dimeric green 
                     fluorescent protein (Hirano et al., 2022)"
                     /label=StayGold
                     /note="engineered variant of a GFP from Cytaeis uchidae"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             complement(2979..3008)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3025..3047
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3062..3086
                     /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     complement(3086..3105)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3087..3105)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3189..3208)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3414..3491
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3492..4574
                     /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     complement(3511..3530)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5383..5574
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5568..5590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5676..5815
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5731..5750
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5830..5847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
       61 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      121 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
      181 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
      241 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
      301 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
      361 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
      421 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
      481 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
      541 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
      601 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
      661 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
      721 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
      781 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
      841 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
      901 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
      961 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     1021 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     1081 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     1141 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     1201 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     1261 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     1321 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     1381 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     1441 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     1501 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     1561 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     1621 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     1681 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1741 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     1801 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     1861 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     1921 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     1981 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     2041 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     2101 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     2161 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     2221 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct cctagggcta
     2281 gctctagact attaggatcc caattgctta agctcgagtt aaagatgagc ttctaacgtc
     2341 tcggactgac agatatgatc gcgttcctcc gtatcatcct tagactgtgt atattgtttg
     2401 cgaatccaat ggtacggtac atctggcgca ggttggttat gaagaggttt gcagatagtg
     2461 ttttgatgtg cttcgacaca ctttgattta tcgctaagca aggggtataa caatgtgacg
     2521 ggacattcaa ctccgtcgtc acgtggaata tgctgcacat cgttcggcaa gctcacattc
     2581 atgtcacctg tcaatacggg gctgttttcc ttgaagtcct ggccagtaaa ctcgacaatg
     2641 ttatgataag tcccattctt catgaagtgt tggtgctttg cgtggattga gccatcacct
     2701 ttgtactgga tgtggcgatc atacgtgaac ccctcgggca tcacctcatg gaaccaattt
     2761 ttcaggcccg acgggtattt tgtatagtat ttcattccgt acccaaacga agtaccaaga
     2821 gccgcccaag acatcggcaa ctttccgctc gtacacacat acttaccttt atgactacct
     2881 tcatgcgagt tcccttcccc ttcaccctcg actgtaaagc ttttcccgtt aattgtcccc
     2941 tttaactgga acttaaacgg cgtgctggcc atagaaccgt gatggtgatg gtgatggtga
     3001 tggtgatggc tgctgcccat tatatctcct tcttaaagtt aaacaaaatt attactagtg
     3061 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
     3121 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     3181 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     3241 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     3301 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     3361 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
     3421 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     3481 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     3541 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     3601 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     3661 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     3721 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     3781 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     3841 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     3901 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     3961 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     4021 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     4081 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     4141 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     4201 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     4261 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     4321 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     4381 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     4441 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     4501 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4561 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     4621 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     4681 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     4741 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     4801 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     4861 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     4921 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     4981 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     5041 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     5101 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     5161 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     5221 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     5281 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     5341 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     5401 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     5461 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     5521 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     5581 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     5641 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     5701 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     5761 accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     5821 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5881 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5941 catgtgagca a
//
