LOCUS       sequence-468949-        5752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the nuclear import substrate NLS-BFP-mEosEM..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5752)
  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 5752)
  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: pTrcHisA-NLS-BFP-mEosEM
FEATURES             Location/Qualifiers
     source          1..5752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..93
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             94..1176
                     /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(113..132)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1408..1413,1414..1430,1431..1437)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1445..1461
                     /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     1450..1472
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    1478..1547
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             1598..1624
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             1643..1663
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1673..2383
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTHGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKIRHNIEDGCVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1700..1728)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2323..2343
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2396..3076
                     /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             3092..3109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3151..3168)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3321..3407
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3499..3526
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3545..3636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3637..3705,3706..4497)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3855..3874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4668..5256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5157..5176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5410..5427
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5442..5582
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5508..5527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5668..5690
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gcatgcattt acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
       61 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
      121 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
      181 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
      241 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
      301 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
      361 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
      421 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
      481 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
      541 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
      601 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
      661 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
      721 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
      781 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
      841 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
      901 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
      961 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     1021 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     1081 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1141 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     1201 gcgcgaattg atctggtttg acagcttatc atcgactgca cggtgcacca atgcttctgg
     1261 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     1321 cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg
     1381 ttctggcaaa tattctgaaa tgagctgttg acaattaatc atccggctcg tataatgtgt
     1441 ggaattgtga gcggataaca atttcacaca ggaaacagcg ccgctgagaa aaagcgaagc
     1501 ggcactgctc tttaacaatt tatcagacaa tctgtgtggg cactcgaccg gaattatcga
     1561 ttaactttat tattaaaaat taaagaggta tatattaatg tatcgattaa ataaggagga
     1621 ataaaccatg agactcgagc ctccaaagaa gaaaagaaag gtcgacagcg cgagcaaagg
     1681 agaagaactc ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     1741 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
     1801 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt
     1861 cactcatggt gttcagtgct tttcaagata cccggatcat atgaaacggc atgacttttt
     1921 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
     1981 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
     2041 gttaaaaggt attgatttta aagaagatgg aaacattctt gggcacaaat tggaatacaa
     2101 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagctaa
     2161 cttcaaaatt agacacaaca ttgaagatgg atgcgttcaa ctagcagacc attatcaaca
     2221 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
     2281 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
     2341 aacagctgct gggattacac atggcatgga tgaactatac aaaggatgcg ctagcatggt
     2401 gagtgcgatt aagccagaca tgaggatcaa actccgtatg gaaggcaacg taaacgggca
     2461 ccactttgtg atcgacggag aaggtacagg caagccttat gagggaaaac agaccatgga
     2521 tcttgaagtc aaagagggcg gacctctgcc ttttgccttt gatatcctga ccactgcatt
     2581 ccattacggc aacagggtat tcgtgaaata tccagacaac atacaagact attttaagca
     2641 gtcgtttcct aaggggtatt cgtgggaacg aagcatgact ttcgaagacg ggggcatttg
     2701 caatgccaga aacgacataa caatggaagg ggacactttc tataataaag ttcgatttta
     2761 tggtaccaac tttcccgcca atggtccagt tatgcagaag aagacgctga aatgggagcc
     2821 ctccactgag aaaatgtatg tgcgtgatgg agtgctgacg ggtgatattg agatggcttt
     2881 gttgcttgaa ggaggtgccc attaccgatg tgacttcaga actacttaca aagctaagga
     2941 gaagggtgtc aagttaccag gcgcccactt tgtggaccac gccattgaga ttttaagcca
     3001 tgacaaagat tacaacaagg ttaagctgta tgagcatgct gttgctcatt ctggattgcc
     3061 tgacaatgcc agacgaggat cctgcgagct ccatcatcat catcatcatt gaaagcttgg
     3121 ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag
     3181 cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
     3241 gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag
     3301 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     3361 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     3421 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     3481 ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     3541 ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3601 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3661 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3721 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3781 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3841 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     3901 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3961 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4021 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4081 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4141 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4201 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4261 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4321 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4381 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4441 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4501 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4561 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4621 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4681 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4741 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4801 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4861 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4921 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4981 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5041 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5101 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5161 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5221 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5281 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5341 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5401 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5461 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     5521 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     5581 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5641 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5701 catcaccgaa acgcgcgagg cagcagatca attcgcgcgc gaaggcgaag cg
//
