LOCUS       sequence-376977-        6456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fusion of hfYFP and Streptavidin core domain separated by a TEV 
            cleavage site for fluorescence-assisted protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6456)
  AUTHORS   Campbell BC, Paez-Segala MG, Looger LL, Petsko GA, Liu CF
  TITLE     Chemically stable fluorescent proteins for advanced microscopy.
  JOURNAL   Nat Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 
            10.1038/s41592-022-01660-7.
  PUBMED    36344833
REFERENCE   2  (bases 1 to 6456)
  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 
            Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 
            10.1038/s41592-022-01660-7."
COMMENT     Sequence Label: pET28a-hfYFP-TEV-SAV
FEATURES             Location/Qualifiers
     source          1..6456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(110..129)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     320..341
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(570..1385)
                     /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(570..1385)
                     /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     1293..1312
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1377..1396
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1507..2095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1996..2015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2249..2266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2281..2420
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2346..2365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2506..2528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2522..2713)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3522..4604)
                     /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     4566..4585
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4605..4682)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4888..4907
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4991..5010
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4991..5009
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5010..5034
                     /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             5049..5071
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5090..5107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5186..5207)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             5894..5914
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6311..6328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6381..6399)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6395..6442
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
       61 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
      121 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
      181 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
      241 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
      301 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
      361 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
      421 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
      481 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      541 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
      601 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
      661 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
      721 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
      781 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
      841 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
      901 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
      961 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     1021 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     1081 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     1141 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     1201 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     1261 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     1321 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     1381 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     1441 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1501 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1561 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1621 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     1681 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1741 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1801 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1861 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1921 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     1981 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2041 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2101 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2161 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2221 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     2281 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     2341 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2401 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2461 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2521 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     2581 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     2641 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     2701 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     2761 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     2821 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     2881 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     2941 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     3001 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     3061 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     3121 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     3181 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     3241 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     3301 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     3361 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     3421 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     3481 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3541 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3601 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     3661 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     3721 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     3781 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     3841 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     3901 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     3961 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4021 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4081 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4141 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4201 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4261 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4321 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4381 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4441 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4501 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4561 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     4621 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     4681 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     4741 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     4801 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     4861 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     4921 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     4981 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     5041 gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca
     5101 tcatcacagt tcaggcaaag cagaaacaat actctctcat atggtgagca agggcgagga
     5161 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     5221 gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagct
     5281 catctccacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttaggcta
     5341 cggcctgatg gtgttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     5401 cgccatgccc gaaggctacg tccaggagcg caccatctct ttcgaggacg acggttacta
     5461 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgtgctgaa
     5521 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa
     5581 cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg ctaacttcaa
     5641 gatccgccac aacgttgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac
     5701 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgt
     5761 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgaaggaga gggtgaccgc
     5821 cgccgggatt acacatgaca tgaacgagct gtacaaggga tccgctggct cagctgctgg
     5881 ttctggtgaa ttcgagaatc tgtattttca gggtcatgct gaagctggta tcaccggtac
     5941 ttggtacaac cagctggggt ctaccttcat cgttaccgct ggtgctgacg gtgcactgac
     6001 cggtacttac gaaagcgctg ttggtaacgc tgaaagccgt tatgttctga ccggtcgtta
     6061 cgactctgct ccggctaccg acggttctgg tactgctctg ggttggaccg ttgcttggaa
     6121 aaacaactac cgtaacgctc actctgctac cacctggtct ggccagtacg ttggtggtgc
     6181 tgaagctcgt atcaacaccc agtggctgct gacctctggt accaccgaag ctaacgcttg
     6241 gaaatctacc ctggttggtc acgacacgtt caccaaagtt aaaccgtctg ctgcttctat
     6301 ctaactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     6361 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     6421 acgggtcttg aggggttttt tgctgaaagg aggaac
//
