LOCUS       sequence-167081-        5681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of transmembrane proteins using N-terminal 
            MISTIC and C-terminal HIS6 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  AUTHORS   Vollmer B, Schooley A, Sachdev R, Eisenhardt N, Schneider AM, 
            Sieverding C, Madlung J, Gerken U, Macek B, Antonin W
  TITLE     Dimerization and direct membrane interaction of Nup53 contribute to 
            nuclear pore complex assembly.
  JOURNAL   EMBO J. 2012 Oct 17;31(20):4072-84. doi: 10.1038/emboj.2012.256. 
            Epub 2012 Sep 7.
  PUBMED    22960634
REFERENCE   2  (bases 1 to 5681)
  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"; doi: 
            "10.1038/emboj.2012.256"; journalName: "EMBO J"; date: "2012-10-17- 
            17"; volume: "31"; issue: "20"; pages: "4072-84"
COMMENT     Sequence Label: pET28a MISTIC
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             352..369
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             373..405
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             455..472
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(525..543)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      539..586
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      623..1078
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(710..729)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     920..941
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1171..1986)
                     /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(1171..1986)
                     /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     1894..1913
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1978..1997
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2108..2696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2597..2616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2850..2867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2882..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     5595..5614
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5595..5613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5614..5638
                     /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             5653..5675
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggccatgt tttgtacatt ttttgaaaaa catcaccgga agtgggacat actgttagaa
       61 aaaagcacgg gtgtgatgga agctatgaaa gtgacgagtg aggaaaagga acagctgagc
      121 acagcaatcg accgaatgaa tgaaggactg gacgcgttta tccagctgta taatgaatcg
      181 gaaattgatg aaccgcttat tcagcttgat gatgatacag ccgagttaat gaagcaggcc
      241 cgagatatgt acggccagga aaagctaaat gagaaattaa atacaattat taaacagatt
      301 ttatccatct cagtatctga agaaggcgaa aaagaaggtt ctggttctgg cctggtgccg
      361 cgcggtagcc atatggctag catgactggt ggacagcaaa tgggtcgcgg atccgaattc
      421 gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
      481 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
      541 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
      601 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
      661 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
      721 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
      781 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
      841 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
      901 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
      961 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     1021 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca
     1081 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1141 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     1201 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     1261 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     1321 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     1381 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     1441 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     1501 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     1561 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     1621 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     1681 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     1741 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     1801 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     1861 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     1921 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     1981 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     2041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2221 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2701 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2761 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2821 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2881 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     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 aggggaattg tgagcggata acaattcccc
     5641 tctagaaata attttgttta actttaagaa ggagatatac c
//
