LOCUS       sequence-167078-        5938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of his6 and SUMO tagged RRM motif of Xenopus 
            laevis Nup53.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5938)
  AUTHORS   Antonin lab
  TITLE     Antonin lab MISTIC plasmid
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5938)
  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            "Unpublished"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: pET28a SUMO Nup53xl 162-267
FEATURES             Location/Qualifiers
     source          1..5938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             13..30
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             40..57
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             64..354
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     CDS             355..375
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             712..729
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(782..800)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      796..843
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      880..1335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(967..986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1177..1198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1428..2243)
                     /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(1428..2243)
                     /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     2151..2170
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2235..2254
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2365..2953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2854..2873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3107..3124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3139..3281
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3207..3226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3367..3389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3383..3574)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4383..5465)
                     /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     5427..5446
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5466..5543)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5749..5768
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5852..5871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5852..5870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5871..5895
                     /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             5910..5932
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
       61 aatatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc agaagtcaag
      121 cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt cttcaagatc
      181 aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa
      241 atggactcct taagattctt gtacgacggt attagaattc aagctgatca gacccctgaa
      301 gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat tggtgagaat
      361 ctttattttc agggccatat ggatcagctg gatgatacct gggttaccgt ttttggtttt
      421 ccgcaggcaa gcgcaagcta tattctgctg cagtttgcac agtatggcaa tattattaaa
      481 catgtgatga gcaataatgg caattggatg catattcagt atcagagcaa actgcaggca
      541 cgtaaagcac tgagcaaaga tggtcgtatt tttggtgaaa gcattatgat tggtgtgaaa
      601 ccgtgcattg ataaaagcgt tatggaagca accgaaaaag ttagcacccc gagcgttagc
      661 agcgttttta cacctccggt taaaagcatt cgtaccccgt gatagctcga gcaccaccac
      721 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      781 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      841 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      901 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
      961 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1021 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1081 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1141 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1201 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1261 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1321 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1381 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     1441 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     1501 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     1561 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     1621 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     1681 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     1741 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     1801 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     1861 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     1921 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     1981 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     2041 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2101 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2161 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2221 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     2281 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3001 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3061 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3121 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3181 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3241 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3301 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3361 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3421 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3481 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3541 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3601 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3661 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3721 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3781 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     3841 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     3901 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     3961 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4021 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4081 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4141 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4201 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4261 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4321 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4441 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4501 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4561 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4621 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     4681 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4741 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4801 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     4861 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     4921 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     4981 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5041 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5101 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5161 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5221 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5281 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5341 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5401 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5461 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5521 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5581 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5641 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     5701 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     5761 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     5821 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     5881 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacc
//
