LOCUS       sequence-331632-        6873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FBF-2 with AS/Y mutations in repeat 5 in E. coli for 
            purification and crystallization studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6873)
  AUTHORS   Bhat VD, McCann KL, Wang Y, Fonseca DR, Shukla T, Alexander JC, Qiu 
            C, Wickens M, Lo TW, Tanaka Hall TM, Campbell ZT
  TITLE     Engineering a conserved RNA regulatory protein repurposes its 
            biological function in vivo.
  JOURNAL   Elife. 2019 Jan 17;8. pii: 43788. doi: 10.7554/eLife.43788.
  PUBMED    30652968
REFERENCE   2  (bases 1 to 6873)
  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: "Elife."; 
            date: "2019-01-17"; volume: "8. pii: 43788. doi"; pages: " 
            10.7554/eLife.43788"
COMMENT     Sequence Label: pSMT3-FBF2 AS/Y
FEATURES             Location/Qualifiers
     source          1..6873
                     /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..450
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             463..483
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1738..1755
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1808..1826)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1822..1869
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1906..2361
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1993..2012)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2203..2224
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2453..3268)
                     /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(2453..3268)
                     /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     3176..3195
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3260..3279
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3390..3978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3879..3898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4132..4149
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4164..4303
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4229..4248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4389..4411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4405..4596)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5405..6487)
                     /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     6449..6468
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6488..6565)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6771..6790
                     /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 gctagcatgt cggactcaga agtcaatcaa
      181 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
      241 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
      301 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
      361 ggtattagaa ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt
      421 attgaggctc acagagaaca gattggtgga tccccggaat tcgaaaacct gtattttcag
      481 ggctcaaaca atgtgctgcc aacctggtcc ttggattcca atggtgaaat gcgttcaaga
      541 ttgtcgctct ctgaagtttt ggatagtggc gatttaatga agtttgccgt tgacaaaact
      601 gggtgccagt tcctagaaaa ggcagtgaag ggcagcctga catcttatca gaagttccaa
      661 ttgtttgaac aagttattgg gagaaaagat gatttcctta aattgagcac gaacatcttt
      721 ggaaattatt tggtacaaag cgttattgga atttcattgg caactaatga tgacggatac
      781 acaaagcgac aagaaaagtt gaagaacttc atcagttccc aaatgacaga tatgtgcttg
      841 gacaaatttg cgtgtcgtgt tatccaatcc agtctccaga acatggattt atcccttgcg
      901 tgtaaacttg ttcaagctct acctcgtgat gctcgtctta tcgcaatctg tgttgatcaa
      961 aatgccaatc acgtcattca gaaagtcgta gcagtcatcc cactgaagaa ttgggagttt
     1021 attgtggatt tcgttgccac cccagagcac ctccgtcaga tttgttctga taagtatggg
     1081 gcctatgttg tgtcaactat tatcgaaaag ctcactgctg attcaatgaa cgttgattta
     1141 acttccgctg ctcaaaatct tcgtgaacga gcgctccaac gactcatgac ttctgtgact
     1201 aatcggtgcc aagaattagc tacgaacgag tatgccaatt acataataca gcatattgta
     1261 tccaatgatg atctggccgt ttatcgggaa tgtatcatag agaaatgcct catgcgcaac
     1321 cttctttcgt tgtcccagga aaagtttgct tctcatgttg ttgagaaagc attcctacat
     1381 gcaccattgg aacttcttgc cgaaatgatg gacgagattt tcgacggata cattccacat
     1441 ccggacacgg gaaaagatgc cctcgatatc atgatgttcc atcaattcgg aaactacgtc
     1501 gttcaatgca tgctgactat ttgctgtgat gctgtgtctg gaagacggca aacgaaagaa
     1561 ggaggctatg accatgcaat ctcattccag gattggctga aaaagttgca ctcgagagtt
     1621 acgaaagaaa ggcatcgctt gtcaaggttc tcatccggca aaaagatgat cgagacgctt
     1681 gccaatctcc gttcaactca tccgatttat gaactccaat aagcggccgc actcgagcac
     1741 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1801 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1861 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1921 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1981 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2041 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2101 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2161 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2221 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2281 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2341 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2401 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2461 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2521 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2581 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2641 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2701 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2761 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2821 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2881 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2941 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     3001 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     3061 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     3121 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     3181 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     3241 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     3301 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4021 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4081 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4141 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4201 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4261 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4321 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4381 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4441 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4501 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4561 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4621 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4681 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4741 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4801 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4861 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4921 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4981 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     5041 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5101 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5161 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5221 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5281 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5341 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5401 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5461 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5521 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5581 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5641 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5701 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5761 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5821 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5881 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5941 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     6001 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     6061 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6121 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6181 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6241 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6301 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6361 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6421 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6481 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6541 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6601 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6661 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6721 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6781 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6841 gcgtagagga tcgagatctc gatcccgcga aat
//
