LOCUS       sequence-315228-        5700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-tagged greenFAST in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5700)
  AUTHORS   Tebo AG, Moeyaert B, Thauvin M, Carlon-Andres I, Boken D, Volovitch 
            M, Padilla-Parra S, Dedecker P, Vriz S, Gautier A
  TITLE     Orthogonal fluorescent chemogenetic reporters for multicolor 
            imaging.
  JOURNAL   Nat Chem Biol. 2020 Aug 10. pii: 10.1038/s41589-020-0611-0. doi: 
            10.1038/s41589-020-0611-0.
  PUBMED    32778846
REFERENCE   2  (bases 1 to 5700)
  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 Chem 
            Biol. 2020 Aug 10. pii: 10.1038/s41589-020-0611-0. doi: 
            10.1038/s41589-020-0611-0."
COMMENT     Sequence Label: pAG261-His-greenFAST
FEATURES             Location/Qualifiers
     source          1..5700
                     /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"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             157..177
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             178..552
                     /codon_start=1
                     /product="small protein tag, derived from photoactive 
                     yellow protein, that allows in vivo fluorescent labeling by
                     addition of a cell-permeant fluorogen (Plamont et al., 
                     2016)"
                     /label=Y-FAST
                     /translation="MEHVAFGSEDIENTLAKMDDEQLDGLAFGAIQLDGDGNILQYNAA
                     EGDITGRDPKQVIGKNFFKDVATGTDSPEFYRKFKEGVASGNLNTMFEWMIPTSRGPTK
                     VKVHMKKALSGDSYWVFVKRV"
     CDS             565..582
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(635..653)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      649..696
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      733..1188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(820..839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1030..1051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1280..2095)
                     /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(1280..2095)
                     /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     2003..2022
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2087..2106
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2217..2805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2706..2725
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2959..2976
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2991..3130
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3056..3075)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3216..3238
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3232..3423)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4232..5314)
                     /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     5276..5295
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5315..5392)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5598..5617
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcgaaa acctgtattt tcagggcatg
      181 gagcatgttg cctttggcag tgaggacatc gagaacactc tggccaaaat ggacgacgaa
      241 caactggatg ggttggcctt tggcgcaatt cagctcgatg gtgacgggaa tatcctgcag
      301 tacaatgctg ctgaaggaga catcacaggc agagatccca aacaggtgat tgggaagaac
      361 ttcttcaagg atgttgcaac tggaacggat tctcccgagt tttaccgcaa attcaaggaa
      421 ggcgtagcgt cagggaatct gaacaccatg ttcgaatgga tgataccgac aagcagggga
      481 ccaaccaagg tcaaggtgca catgaagaaa gccctttccg gtgacagcta ttgggtcttt
      541 gtgaaacggg tgtaatagct cgagcaccac caccaccacc actgagatcc ggctgctaac
      601 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
      661 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
      721 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      961 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1021 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1081 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1141 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1201 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1261 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1321 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1381 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1441 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     1501 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     1561 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     1621 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     1681 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1741 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1801 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1861 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1921 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1981 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2041 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2101 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2161 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2221 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2281 gtggtttgtt tgccggatca agagctacca gctctttttc cgaaggtaac tggcttcagc
     2341 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2401 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2461 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2521 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2581 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2641 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2701 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2761 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2821 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2881 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2941 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3001 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3061 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3121 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3181 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3241 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3301 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3361 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3421 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     3481 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     3541 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     3601 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     3661 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     3721 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     3781 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     3841 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     3901 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     3961 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4021 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4081 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4141 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4201 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4261 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4321 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4381 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     4441 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     4501 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     4561 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     4621 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     4681 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     4741 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     4801 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     4861 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     4921 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     4981 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5041 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5101 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5161 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5221 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5281 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5341 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     5401 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     5461 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     5521 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     5581 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     5641 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
