LOCUS       sequence-254929-        5700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-tagged iFAST in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5700)
  AUTHORS   Tebo AG, Pimenta FM, Zhang Y, Gautier A
  TITLE     Improved Chemical-Genetic Fluorescent Markers for Live Cell 
            Microscopy.
  JOURNAL   Biochemistry. 2018 Oct 2;57(39):5648-5653. doi: 
            10.1021/acs.biochem.8b00649. Epub 2018 Sep 17.
  PUBMED    30204425
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"; doi: 
            "10.1021/acs.biochem.8b00649"; journalName: "Biochemistry"; date: 
            "2018-10-2- 2"; volume: "57"; issue: "39"; pages: "5648-5653"
COMMENT     Sequence Label: pAG247 His-iFAST
FEATURES             Location/Qualifiers
     source          1..5700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     520..539
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(559..636)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     842..861
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     945..964
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        945..963
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    964..988
                     /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             1003..1025
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1044..1061
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1071..1088
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1101..1121
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1122..1496
                     /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="MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTK
                     VKIHMKKALSGDSYWVFVKRV"
     CDS             1509..1526
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1579..1597)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1593..1640
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1677..2132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1764..1783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1974..1995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2224..3039)
                     /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(2224..3039)
                     /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     2947..2966
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3031..3050
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3161..3749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3650..3669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3903..3920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3935..4074
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4000..4019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4160..4182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4176..4367)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5176..5700,1..558))
                     /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"
ORIGIN
        1 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
       61 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
      121 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
      181 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
      241 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
      301 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
      361 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
      421 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
      481 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
      541 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
      601 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
      661 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
      721 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
      781 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
      841 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
      901 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
      961 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1021 ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc ctggtgccgc
     1081 gcggcagcca tatggctagc gaaaacctgt attttcaggg catggagcat gttgcctttg
     1141 gcagtgagga catcgagaac actctggcca aaatggacga cggacaactg gatgggttgg
     1201 cctttggcgc aattcagctc gatggtgacg ggaatatcct gcagtacaat gctgctgaag
     1261 gagacatcac aggcagagat cccaaacagg tgattgggaa gaacttcttc aaggatgttg
     1321 cacctggaac ggattctccc gagttttacg gcaaattcaa ggaaggcgta gcgtcaggga
     1381 atctgaacac catgttcgaa tggatgatac cgacaagcag gggaccaacc aaggtcaaga
     1441 tacacatgaa gaaagccctt tccggtgaca gctattgggt ctttgtgaaa cgggtgtaat
     1501 agctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1561 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1621 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1681 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1741 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1801 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1861 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1921 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1981 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2041 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2101 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2161 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     2221 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     2281 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     2341 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     2401 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     2461 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     2521 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     2581 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     2641 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     2701 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     2761 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     2821 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2881 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2941 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     3001 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     3061 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3121 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3181 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3241 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3301 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3361 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3421 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3481 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3541 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3601 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3661 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3721 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3781 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3841 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3901 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3961 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4021 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4081 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4141 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4201 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4261 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4321 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4381 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4441 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4501 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4561 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4621 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4681 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4741 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4801 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4861 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4921 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4981 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     5041 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     5101 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5161 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5221 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5281 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5341 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5401 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5461 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5521 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5581 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5641 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
//
