LOCUS       sequence-441662-        7127 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express DDX53 mutant K272A protein in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7127)
  TITLE     DDX53 and 3 x FLAG
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7127)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET29a-DDX53-K272A
FEATURES             Location/Qualifiers
     source          1..7127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2064..2086
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2101..2125
                     /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)."
     primer_bind     complement(2125..2144)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2126..2144)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2232..2251)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2457..2534
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2535..3617
                     /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     complement(2554..2573)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4426..4617
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4611..4633)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4719..4858
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4774..4793
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4873..4890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5044..5632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5124..5143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5743..5762)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5754..6569
                     /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             5754..6569
                     /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     complement(5827..5846)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6661..7116)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6798..6819)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7010..7029
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagacttaaa aaataaaact
      181 ccttacgtct ttgtccaggt tctcttgatc gtgtttctct gtgcctcttt tgttgattta
      241 acttgtattg ctcagccatt actacaagat cttccggaac actctgattt gctctgtcca
      301 gaattttaat caattcaccg gccattttcg aatctctctg agtgatgagg gtaactgatg
      361 tgccagtctt tcctgtccgt ccaatgtacc ctactctgtg tacatatacg tcaatattcc
      421 ttgggaaatc ataattatat acatgtgtga catcattaag atcaagacct cgggatacta
      481 tatcagttgt aatcagtatc tttatgtttc cgcttttaaa gtcctctact gctcgctctt
      541 gatcactctg ttcactgttg ccatgtaatg attctgcaga tatgccttgg atattgaagt
      601 cgcttgacaa gtcatcagca atatgttttt ggctgacaaa catgatgact ttgtcgttgg
      661 gtgacatgtt ctctacgaat tcttgggtga gagctcgttt ttctttttct gtggtaacaa
      721 ttatattttg cttcactgta tttacagcca ctagattcag attaccaaca taaacaatca
      781 taggatcttt caaataagaa agtgctagtt gacgtacagt atctggccaa gttgcacttg
      841 tcataacagt ctgtcggtct gggcgcacat ctaataaaat cttccttatc tggggttcaa
      901 attccatatc cagcatttta tctgcctcat ctataaccaa gtaggttatg cttcttaggt
      961 tgacagagtt attcatttgt aggtcattca gcctcccagg agttgcaata atgatatcta
     1021 cacctttgct aatgtcttct atttgtccat ttctgtttct accaccatat atgcaaatgc
     1081 ttttgagacc tttatatgaa tactttgaac attcagcttc cacgtgaaga gccaactctc
     1141 tagtgggtgt aaggactagc atcccaggcc cattcctttg ctctctagat attggttgag
     1201 aatcaagatg aataaaccca ggcattagat aggacaatgt tgcccctgtt ccggtttgtg
     1261 caactactat aagatctatt ccttgtagaa taattggcca tgcctgtgac tgaattggcg
     1321 ttggctttaa aatccctacc cttattatgc ttttcagaag atcagggtat tgctgaaaag
     1381 cgtctttaaa cctgcaagtt ggttttggaa tgagacgctt ttcaccactt ttcaagtcat
     1441 cacacgttat gttgaaattt tcctttctcc agttaatcac ctgcatttca gacatgcagc
     1501 ttgttgcttt ggattctatg taaaagtttt tcttaactgg tggtagatct gcccattttc
     1561 tcttttcgca ctccacgact gctgccctaa tgcgatccca atttgacaat ggctcagctt
     1621 ctccaacaat gtcatttctg cctagatttc ttccaatagg ggtttgggat gcagcattat
     1681 ccacactgga ttctgagttg tagctttctt gttttctaat aagtgtttct atagccgctt
     1741 tggcctttgc tttcatttcc ctattgccaa aaattctgac ttttgcttca gattccccgt
     1801 ttatgatctg tattttagtg tttgtcgaat gttgtagatc ttttattttt gatccactgt
     1861 aaccaatgac cacaccaacc atattgttct ttattttaaa gcagagtggt ggttcacggg
     1921 agcctgctgc tctcggtccc tgatggccga aggggccact ccagccactg cctctgctgc
     1981 ccctgacatc ccagctggcc ccaaggtctc ttggattagc ctccgccctc ttccactctg
     2041 gggcccagtg ggacatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
     2101 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     2161 atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     2221 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     2281 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     2341 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     2401 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     2461 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     2521 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     2581 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     2641 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     2701 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     2761 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     2821 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     2881 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     2941 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     3001 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     3061 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     3121 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     3181 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     3241 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     3301 ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata
     3361 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     3421 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     3481 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     3541 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3601 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     3661 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     3721 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     3781 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     3841 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     3901 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     3961 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     4021 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     4081 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     4141 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     4201 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     4261 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     4321 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     4381 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     4441 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     4501 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     4561 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4621 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4681 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     4741 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     4801 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     4861 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4921 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4981 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5041 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5101 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5161 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5221 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5281 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5341 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5401 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5461 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5521 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5581 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5641 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5701 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     5761 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     5821 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     5881 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     5941 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     6001 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     6061 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     6121 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     6181 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     6241 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     6301 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     6361 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     6421 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     6481 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     6541 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     6601 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6661 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6721 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     6781 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6841 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     6901 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6961 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7021 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7081 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
