LOCUS       sequence-330608-        5613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human TBP6.9  with the F34M/F37M/F77M triple mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5613)
  AUTHORS   Srivastava Y, Bonn-Breach R, Chavali SS, Lippa GM, Jenkins JL, 
            Wedekind JE
  TITLE     Affinity and Structural Analysis of the U1A RNA Recognition Motif 
            with Engineered Methionines to Improve Experimental Phasing.
  JOURNAL   Crystals (Basel). 2021 Mar;11(3). doi: 10.3390/cryst11030273. Epub 
            2021 Mar 10.
  PUBMED    33777416
REFERENCE   2  (bases 1 to 5613)
  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: "Crystals 
            (Basel)."; date: "2021-03"; volume: "11(3). doi"; pages: " 
            10.3390/cryst11030273. Epub 2021 Mar 10"
COMMENT     Sequence Label: pET28a-TEV-TBP6.9F34MF37MF77M
FEATURES             Location/Qualifiers
     source          1..5613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..128
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             459..476
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(529..547)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      543..590
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      627..1082
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(714..733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     924..945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1174..1989)
                     /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(1174..1989)
                     /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     1897..1916
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1981..2000
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2111..2699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2600..2619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2853..2870
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2885..3024
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2950..2969)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3110..3132
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3126..3317)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4126..5208)
                     /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     5170..5189
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5209..5286)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5492..5511
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5595..5613,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5595..5613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcgag aatctttatt
      121 ttcagggcca tatggcagtt cccgagaccc gccctaacca cactatttat atcaacaacc
      181 tcaattcgaa gatcaagaag gatgagctaa aaaagtccct gcacgccatc atgtcccgga
      241 tgggccagat cctggatatc ctggtaccgc ggcgccgtac gccgaggggc caggcctttg
      301 tcatcttcaa ggaggtcagc agcgccacca acgccctgcg ctccatgcag ggtttcccta
      361 tgtatgacaa acctatgcgt atccagtatg ccaagaccga caagcgtatc ccggccaaga
      421 tgaaatgata ggtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
      481 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
      541 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
      601 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
      661 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      721 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      781 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      841 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      901 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      961 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1021 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     1081 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1141 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     1201 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     1261 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     1321 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1381 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     1441 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     1501 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     1561 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1621 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     1681 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     1741 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     1801 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     1861 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     1921 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     1981 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     2041 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2221 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2821 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2881 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     2941 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3001 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3061 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3121 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3181 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3241 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3301 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3361 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3421 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3481 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3541 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3601 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3661 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     3721 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     3781 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     3841 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     3901 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     3961 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4021 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4081 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4141 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4201 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4261 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4321 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4381 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4441 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4501 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     4561 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4621 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     4681 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     4741 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     4801 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     4861 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     4921 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     4981 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5041 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5101 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5161 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5221 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5281 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5341 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5401 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5461 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     5521 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     5581 cgatcccgcg aaattaatac gactcactat agg
//
