LOCUS       Exported                5697 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses His-tagged FAST (also called YFAST) in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    pAG87 FAST
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5697)
  AUTHORS   Plamont MA, Billon-Denis E, Maurin S, Gauron C, Pimenta FM, Specht 
            CG, Shi J, Querard J, Pan B, Rossignol J, Moncoq K, Morellet N, 
            Volovitch M, Lescop E, Chen Y, Triller A, Vriz S, Le Saux T, Jullien
            L, Gautier A
  TITLE     Small fluorescence-activating and absorption-shifting tag for 
            tunable protein imaging in vivo.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jan 19;113(3):497-502. doi: 
            10.1073/pnas.1513094113. Epub 2015 Dec 28.
  PUBMED    26711992
REFERENCE   2  (bases 1 to 5697)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5697
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     54..73
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     157..176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        157..175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    176..200
                     /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             215..237
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             256..273
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             283..300
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             313..333
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             334..705
                     /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="EHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAE
                     GDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTKV
                     KVHMKKALSGDSYWVFVKRV"
     CDS             718..735
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(788..806)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      802..849
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      886..1341
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(973..992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1183..1204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1433..2248)
                     /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     2156..2175
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2240..2259
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2370..2958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2859..2878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3112..3129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3144..3283
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3209..3228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3369..3391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3385..3576)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4385..5467)
                     /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     5429..5448
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5468..5545)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
       61 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
      121 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
      181 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
      241 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
      301 catatggcta gcgaaaacct gtattttcag ggcgaacatg ttgcctttgg tagcgaagat
      361 atcgagaata ctctagcgaa aatggatgat ggccaattag atggattagc ctttggtgct
      421 atacagcttg atggtgatgg caatattctg cagtataatg cagctgaagg agacataaca
      481 gggagagatc ccaaacaagt gattggcaag aacttcttca aagacgtagc accaggtaca
      541 gattctcctg aattttacgg gaaattcaag gaaggagtag catcaggtaa cttgaatacc
      601 atgttcgagt ggatgattcc gacgagtagg gggccaacca aggttaaagt ccacatgaag
      661 aaggctttga gtggtgactc ctattgggtg tttgtcaaaa gggtttaata gctcgagcac
      721 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      781 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      841 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      901 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      961 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1021 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1081 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1141 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1201 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1261 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1321 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1381 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1441 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1501 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1561 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1621 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1681 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1741 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1801 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1861 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1921 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1981 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2041 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2101 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2161 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2221 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2281 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2341 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2401 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2461 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2521 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2581 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2641 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2701 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2761 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2821 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2881 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2941 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3001 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3061 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3121 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3181 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3241 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3301 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3361 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3421 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3481 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3541 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3601 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3661 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     3721 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     3781 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     3841 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     3901 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     3961 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4021 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4081 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4141 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4201 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4261 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4321 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4381 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4441 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4501 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4561 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4621 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     4681 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4741 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4801 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4861 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4921 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     4981 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5041 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5101 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5161 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5221 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5281 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5341 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5401 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5461 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5521 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5581 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5641 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac cataccc
//
