LOCUS       sequence-386362-        7291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of artificial transmembrane protein tagged with mEGFP and
            MBP at the plasma membrane. Control construct for single molecule 
            co-localization and co-tracking microscopy..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7291)
  AUTHORS   Sotolongo Bellon J, Birkholz O, Richter CP, Eull F, Kenneweg H, 
            Wilmes S, Rothbauer U, You C, Walter MR, Kurre R, Piehler J
  TITLE     Four-color single-molecule imaging with engineered tags resolves the
            molecular architecture of signaling complexes in the plasma 
            membrane.
  JOURNAL   Cell Rep Methods. 2022 Feb 4;2(2):100165. doi: 
            10.1016/j.crmeth.2022.100165. eCollection 2022 Feb 28.
  PUBMED    35474965
REFERENCE   2  (bases 1 to 7291)
  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: "Cell Rep 
            Methods."; date: "2022-02-4"; volume: "2(2):100165. doi"; pages: " 
            10.1016/j.crmeth.2022.100165. eCollection 2022 Feb 28"
COMMENT     Sequence Label: pSems-leader-mEGFP-MBP-TMD(ALA7)
FEATURES             Location/Qualifiers
     source          1..7291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     918..977
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             981..1007
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1017..1019,1020..1022,1023..1733)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1062..1083)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1323..1342)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1670..1691
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1749..2849
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2823..2846
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2898..2909
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     intron          3068..3297
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3280..3299
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3369..3943,3944..3946,3947..3955)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3536..3553)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3763..3782
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3978..4781
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4041..4060)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4651..4670
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4911..5032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4948..4967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5134..5150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5134..5150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5147..5169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5158..5174
                     /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)."
     promoter        complement(join(5182..5188,5189..5206,5207..5212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5227..5248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5365..5382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5536..6124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5616..5635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6295..7086,7087..7155))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6918..6937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7156..7260)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatcatg gagacagaca cactcctgct atgggtactg ctgctctggg
      961 ttccaggttc cactggtgac tatccatatg atgttccaga ttatgctgaa ttcaccatgg
     1021 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1081 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1141 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1201 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1261 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1321 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1381 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1441 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1501 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1561 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1621 tgagcaccca gtccaaactg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1681 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggatcca
     1741 tgtggaatat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     1801 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     1861 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     1921 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     1981 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     2041 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     2101 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     2161 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     2221 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     2281 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     2341 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     2401 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     2461 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     2521 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     2581 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     2641 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     2701 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     2761 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     2821 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     2881 ataacaacaa cctcgggatc gagggaagga tttcaaccgg tgcactggca gcactggcag
     2941 cgctggcagc cctggcggca ctggcagcac tggcagcact ggcaaaaagc tctagataag
     3001 cggccgcata gataactgat ccagtgtgct ggaattaatt cgctgtctgc gagggccagc
     3061 tgttggggtg agtactccct ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt
     3121 tccaaaaacg aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc
     3181 gcgtccatct ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt
     3241 gagatctggc catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg
     3301 tccactccca ggtccaactg caggtcgagc atgcatctag ggcggccaat tccgcccctc
     3361 tccccccccc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa
     3421 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     3481 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     3541 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     3601 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     3661 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     3721 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     3781 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     3841 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     3901 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aagcttgcca caacccggga
     3961 taattcctgc agccaatatg ggatcggcca ttgaacaaga tggattgcac gcaggttctc
     4021 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4081 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4141 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     4201 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4261 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4321 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4381 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4441 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4501 ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt cgtgacccat ggcgatgcct
     4561 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4621 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4681 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4741 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aggggatcaa ttctctagat
     4801 aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     4861 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     4921 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4981 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtcg
     5041 agtgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5101 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5161 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5221 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5281 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5341 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5401 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5461 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5521 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5581 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5641 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5701 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5761 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5821 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5881 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5941 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6001 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6061 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6121 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6181 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6241 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6301 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6361 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6421 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6481 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6541 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6601 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6661 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6721 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6781 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6841 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6901 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6961 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7021 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7081 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7141 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7201 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7261 cacatttccc cgaaaagtgc cacctgacgt c
//
