LOCUS       sequence-466940-        8553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ImpBeta1 (human) with mEosEM fused at the C-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8553)
  AUTHORS   Sau A, Schnorrenberg S, Huang Z, Bandyopadhyay D, Sharma A, Gurth 
            CM, Dave S, Musser SM
  TITLE     Overlapping nuclear import and export paths unveiled by two-colour 
            MINFLUX.
  JOURNAL   Nature. 2025 Mar 19. doi: 10.1038/s41586-025-08738-0.
  PUBMED    40108461
REFERENCE   2  (bases 1 to 8553)
  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: "Nature. 
            2025 Mar 19. doi: 10.1038/s41586-025-08738-0."
COMMENT     Sequence Label: pET28a-ImpBeta1-mEosEM
FEATURES             Location/Qualifiers
     source          1..8553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2335..2354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2495..2517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2511..2702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3511..4593)
                     /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     4555..4574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4594..4671)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4877..4896
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4980..4999
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4980..4998
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4999..5023
                     /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             5038..5060
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7701..8381
                     /codon_start=1
                     /product="bright monomeric photocontrollable fluorescent 
                     protein that withstands Epon embedding and osmium tetroxide
                     treatment (Fu et al., 2020)"
                     /label=mEosEM
                     /note="shows on and off switching in the green form after 
                     Epon embedding"
                     /translation="MVSAIKPDMRIKLRMEGNVNGHHFVIDGEGTGKPYEGKQTMDLEV
                     KEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSMTFEDGGICNA
                     RNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALL
                     LEGGAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLP
                     DNARR"
     CDS             8397..8414
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8467..8485)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8481..8528
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3781 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     4981 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     5041 gtttaacttt aagaaggaga tataccatgg agctgatcac cattctcgag aagaccgtgt
     5101 ctcccgatcg gctggagctg gaagcggcgc agaagttcct ggagcgtgcg gccgtggaga
     5161 acctgcccac tttccttgtg gaactgtcca gagtgctggc aaatccagga aacagtcagg
     5221 ttgccagagt tgcagctggt ctacaaatca agaactcttt gacatctaaa gatccagata
     5281 tcaaggcaca atatcagcag aggtggcttg ctattgatgc taatgctcga cgagaagtca
     5341 agaactatgt tttgcagaca ttgggtacag aaacttaccg gcctagttct gcctcacagt
     5401 gtgtggctgg tattgcttgt gcagagatcc cagtaaacca gtggccagaa ctcattcctc
     5461 agctggtggc caatgtcaca aaccccaaca gcacagagca catgaaggag tcgacattgg
     5521 aagccatcgg ttatatttgc caagatatag acccagagca gctacaagat aaatccaatg
     5581 agattctgac tgccataatc caggggatga ggaaagaaga gcctagtaat aatgtgaagc
     5641 tagctgctac gaatgcactc ctgaactcat tggagttcac caaagcaaac tttgataaag
     5701 agtctgaaag gcactttatt atgcaggtgg tctgtgaagc cacacagtgt ccagatacga
     5761 gggtacgagt ggctgcttta cagaatctgg tgaagataat gtccttatat tatcagtaca
     5821 tggagacata tatgggtcct gctctttttg caatcacaat cgaagcaatg aaaagtgaca
     5881 ttgatgaggt ggctttacaa gggatagaat tctggtccaa tgtctgtgat gaggaaatgg
     5941 atttggccat tgaagcttca gaggcagcag aacaaggacg gccccctgag cacaccagca
     6001 agttttatgc gaagggagca ctacagtatc tggttccaat cctcacacag acactaacta
     6061 aacaggacga aaatgatgat gacgatgact ggaacccctg caaagcagca ggggtgtgcc
     6121 tcatgcttct ggccacctgc tgtgaagatg acattgtccc acatgtcctc cccttcatta
     6181 aagaacacat caagaaccca gattggcggt accgggatgc agcagtgatg gcttttggtt
     6241 gtatcttgga aggaccagag cccagtcagc tcaaaccact agttatacag gctatgccca
     6301 ccctaataga attaatgaaa gaccccagtg tagttgttcg agatacagct gcatggactg
     6361 taggcagaat ttgtgagctg cttcctgaag ctgccatcaa tgatgtctac ttggctcccc
     6421 tgctacagtg tctgattgag ggtctcagtg ctgaacccag agtggcttca aatgtgtgct
     6481 gggctttctc cagtctggct gaagctgctt atgaagctgc agacgttgct gatgatcagg
     6541 aagaaccagc tacttactgc ttatcttctt catttgaact catagttcag aagctcctag
     6601 agactacaga cagacctgat ggacaccaga acaacctgag gagttctgca tatgaatctc
     6661 tgatggaaat tgtgaaaaac agtgccaagg attgttatcc tgctgtccag aaaacgactt
     6721 tggtcatcat ggaacgactg caacaggttc ttcagatgga gtcacatatc cagagcacat
     6781 ccgatagaat ccagttcaat gaccttcagt ctttactctg tgcaactctt cagaatgttc
     6841 ttcggaaagt gcaacatcaa gatgctttgc agatctctga tgtggttatg gcctccctgt
     6901 taaggatgtt ccaaagcaca gctgggtctg ggggagtaca agaggatgcc ctgatggcag
     6961 ttagcacact ggtggaagtg ttgggtggtg aattcctcaa gtacatggag gcctttaaac
     7021 ccttcctggg cattggatta aaaaattatg ctgaatacca ggtttgtttg gcagctgtgg
     7081 gcttagtggg agacttgtgc cgtgccctgc aatccaacat catacctttc tgtgacgagg
     7141 tgatgcagct gcttctggaa aatttgggga atgagaacgt ccacaggtct gtgaagccgc
     7201 agattctgtc agtgtttggt gatattgccc ttgctattgg aggagagttt aaaaaatact
     7261 tagaggttgt attgaatact cttcagcagg cctcccaagc ccaggtggac aagtcagact
     7321 atgacatggt ggattatctg aatgagctaa gggaaagctg cttggaagcc tatactggaa
     7381 tcgtccaggg attaaagggg gatcaggaga acgtacaccc ggatgtgatg ctggtacaac
     7441 ccagagtaga atttattctg tctttcattg accacattgc tggagatgag gatcacacag
     7501 atggagtagt agcttgtgct gctggactaa taggggactt atgtacagca tttgggaagg
     7561 atgtactgaa attagtagaa gctaggccaa tgatccatga attgttaact gaagggcgga
     7621 gatcgaagac taacaaagca aaaacccttg ctacatgggc aacaaaagaa ctgaggaaac
     7681 tgaagaacca agctcaattg atggtgagtg cgattaagcc agacatgagg atcaaactcc
     7741 gtatggaagg caacgtaaac gggcaccact ttgtgatcga cggagaaggt acaggcaagc
     7801 cttatgaggg aaaacagacc atggatcttg aagtcaaaga gggcggacct ctgccttttg
     7861 cctttgatat cctgaccact gcattccatt acggcaacag ggtattcgtg aaatatccag
     7921 acaacataca agactatttt aagcagtcgt ttcctaaggg gtattcgtgg gaacgaagca
     7981 tgactttcga agacgggggc atttgcaatg ccagaaacga cataacaatg gaaggggaca
     8041 ctttctataa taaagttcga ttttatggta ccaactttcc cgccaatggt ccagttatgc
     8101 agaagaagac gctgaaatgg gagccctcca ctgagaaaat gtatgtgcgt gatggagtgc
     8161 tgacgggtga tattgagatg gctttgttgc ttgaaggagg tgcccattac cgatgtgact
     8221 tcagaactac ttacaaagct aaggagaagg gtgtcaagtt accaggcgcc cactttgtgg
     8281 accacgccat tgagatttta agccatgaca aagattacaa caaggttaag ctgtatgagc
     8341 atgctgttgc tcattctgga ttgcctgaca atgccagacg agcggccgca ctcgagcacc
     8401 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     8461 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     8521 gttttttgct gaaaggagga actatatccg gat
//
