LOCUS       sequence-376971-        6627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fusion of hfYFP and Bacillus circulans xylanase 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 6627)
  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 6627)
  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-Bcx
FEATURES             Location/Qualifiers
     source          1..6627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(114..133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     324..345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(574..1389)
                     /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(574..1389)
                     /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     1297..1316
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1381..1400
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1511..2099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2000..2019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2253..2270
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2285..2424
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2350..2369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2510..2532
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2526..2717)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3526..4608)
                     /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     4570..4589
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4609..4686)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4892..4911
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4995..5014
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4995..5013
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5014..5038
                     /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             5053..5075
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5094..5111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5190..5211)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             5898..5918
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6486..6503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6556..6574)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6570..6617
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
       61 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      121 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      181 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      241 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      301 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      361 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      421 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      481 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      541 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
      601 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
      661 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
      721 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
      781 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
      841 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
      901 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
      961 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1021 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     1081 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     1141 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     1201 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     1261 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     1321 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     1381 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     1441 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1501 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1561 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1621 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     1681 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1741 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1801 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1861 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1921 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1981 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2041 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2101 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2161 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2221 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2281 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     2341 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     2401 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     2461 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2521 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     2581 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     2641 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     2701 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     2761 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     2821 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     2881 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     2941 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3001 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3061 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     3121 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     3181 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     3241 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     3301 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     3361 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     3421 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     3481 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     3541 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3601 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     3661 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     3721 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     3781 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     3841 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     3901 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     3961 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4021 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     4081 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     4141 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     4201 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     4261 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     4321 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     4381 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     4441 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     4501 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     4561 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     4621 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     4681 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     4741 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     4801 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     4861 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     4921 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     4981 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     5041 tctagaaata attttgttta actttaagaa ggagatatac catgggcagc agccatcatc
     5101 atcatcatca cagttcaggc aaagcagaaa caatactctc tcatatggtg agcaagggcg
     5161 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     5221 acaagttcag cgtccgcggc gagggcgagg gcgatgccac caacggcaag ctgaccctga
     5281 agctcatctc caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttag
     5341 gctacggcct gatggtgttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
     5401 agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctctttcgag gacgacggtt
     5461 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgtgc
     5521 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     5581 tcaacagcca caacgtctat atcacggccg acaagcagaa gaacggcatc aaggctaact
     5641 tcaagatccg ccacaacgtt gaggacggcg gcgtgcagct cgccgaccac taccagcaga
     5701 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     5761 ccgtcctgag caaagacccc aacgagaagc gcgatcacat ggtcctgaag gagagggtga
     5821 ccgccgccgg gattacacat gacatgaacg agctgtacaa gggatccgct ggctcagctg
     5881 ctggttctgg tgaattcgag aatctgtatt ttcagggtca tgctagcaca gattactggc
     5941 aaaactggac agacggtggc ggtatcgtta atgccgtgaa cggctccgga ggcaactaca
     6001 gcgtgaattg gtctaatact gggaacttcg tagtcggaaa aggttggacg acaggatccc
     6061 cgttccgtac gatcaactac aacgctggcg tttgggcccc gaatggtaac ggttacctga
     6121 cactgtatgg ctggacgcgt tcgccactga ttgaatatta cgttgtcgac tcttggggaa
     6181 cgtaccgtcc gactggaacc tacaaaggca cagtcaaaag cgatggtggt acctatgaca
     6241 tctacaccac cacaagatac aacgcacctt ccatcgatgg cgatcggacc acctttactc
     6301 agtattggag tgttagacaa tctaagcggc cgactggttc gaacgccacc attacgttca
     6361 ccaatcacgt gaatgcatgg aaatcccacg gtatgaacct aggttctaat tgggcttatc
     6421 aagtaatggc gaccgaaggc taccagagct ctggttcttc caacgttaca gtgtggtaac
     6481 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     6541 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     6601 tcttgagggg ttttttgctg aaaggag
//
