LOCUS       sequence-457424-        7913 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of TPOR with ALFAtag and monomeric XFP in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7913)
  AUTHORS   Winkelmann H, Richter CP, Eising J, Piehler J, Kurre R
  TITLE     Correlative single-molecule and structured illumination microscopy 
            of fast dynamics at the plasma membrane.
  JOURNAL   Nat Commun. 2024 Jul 10;15(1):5813. doi: 10.1038/s41467-024-49876-9.
  PUBMED    38987559
REFERENCE   2  (bases 1 to 7913)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-07-10"; volume: "15(1):5813. doi"; pages: " 
            10.1038/s41467-024-49876-9"
COMMENT     Sequence Label: pSems leader-ALFA-mXFP-TpoR
FEATURES             Location/Qualifiers
     source          1..7913
                     /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..980
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     CDS             981..1007
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1017..1055
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             join(1068..1070,1071..1073,1074..1784)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTFGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1113..1134)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1374..1393)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1721..1742
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          3689..3918
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3901..3920
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3991..4565,4566..4568,4569..4577)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4158..4175)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4385..4404
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4600..5403
                     /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(4663..4682)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5273..5292
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5533..5654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5570..5589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5756..5772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5756..5772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5769..5791)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5780..5796
                     /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(5804..5810,5811..5828,5829..5834))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5849..5870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5987..6004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6158..6746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6238..6257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6917..7708,7709..7777))
                     /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     7540..7559
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7778..7882)
                     /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 ttcccttcaa
     1021 gactggaaga agaacttcga aggagactga cagaacccga attcaccatg gtgagcaagg
     1081 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1141 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1201 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1261 tgacctttgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1321 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1381 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1441 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1501 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1561 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1621 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1681 agtccaaact gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1741 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagaccggt caagatgtct
     1801 ccttgctggc atcagactca gagcccctga agtgtttctc ccgaacattt gaggacctca
     1861 cttgcttctg ggatgaggaa gaggcagcgc ccagtgggac ataccagctg ctgtatgcct
     1921 acccgcggga gaagccccgt gcttgccccc tgagttccca gagcatgccc cactttggaa
     1981 cccgatacgt gtgccagttt ccagaccagg aggaagtgcg tctcttcttt ccgctgcacc
     2041 tctgggtgaa gaatgtgttc ctaaaccaga ctcggactca gcgagtcctc tttgtggaca
     2101 gtgtaggcct gccggctccc cccagtatca tcaaggccat gggtgggagc cagccagggg
     2161 aacttcagat cagctgggag gagccagctc cagaaatcag tgatttcctg aggtacgaac
     2221 tccgctatgg ccccagagat cccaagaact ccactggtcc cacggtcata cagctgattg
     2281 ccacagaaac ctgctgccct gctctgcaga ggcctcactc agcctctgct ctggaccagt
     2341 ctccatgtgc tcagcccaca atgccctggc aagatggacc aaagcagacc tccccaagta
     2401 gagaagcttc agctctgaca gcagagggtg gaagctgcct catctcagga ctccagcctg
     2461 gcaactccta ctggctgcag ctgcgcagcg aacctgatgg gatctccctc ggtggctcct
     2521 ggggatcctg gtccctccct gtgactgtgg acctgcctgg agatgcagtg gcacttggac
     2581 tgcaatgctt taccttggac ctgaagaatg ttacctgtca atggcagcaa caggaccatg
     2641 ctagctccca aggcttcttc taccacagca gggcacggtg ctgccccaga gacaggtacc
     2701 ccatctggga gaactgcgaa gaggaagaga aaacaaatcc aggactacag accccacagt
     2761 tctctcgctg ccacttcaag tcacgaaatg acagcattat tcacatcctt gtggaggtga
     2821 ccacagcccc gggtactgtt cacagctacc tgggctcccc tttctggatc caccaggctg
     2881 tgcgcctccc caccccaaac ttgcactgga gggagatctc cagtgggcat ctggaattgg
     2941 agtggcagca cccatcgtcc tgggcagccc aagagacctg ttatcaactc cgatacacag
     3001 gagaaggcca tcaggactgg aaggtgctgg agccgcctct cggggcccga ggagggaccc
     3061 tggagctgcg cccgcgatct cgctaccgtt tacagctgcg cgccaggctc aacggcccca
     3121 cctaccaagg tccctggagc tcgtggtcgg acccaactag ggtggagacc gccaccgaga
     3181 ccgcctggat ctccttggtg accgctctgc atctagtgct gggcctcagc gccgtcctgg
     3241 gcctgctgct gctgaggtgg cagtttcctg cacactacag gagactgagg catgccctgt
     3301 ggccctcact tccagacctg caccgggtcc taggccagta ccttagggac actgcagccc
     3361 tgagcccgcc caaggccaca gtctcagata cctgtgaaga agtggaaccc agcctccttg
     3421 aaatcctccc caagtcctca gagaggactc ctttgcccct gtgttcctcc caggcccaga
     3481 tggactaccg aagattgcag ccttcttgcc tggggaccat gcccctgtct gtgtgcccac
     3541 ccatggctga gtcagggtcc tgctgtacca cccacattgc caaccattcc tacctaccac
     3601 taagctattg gcagcagcct gcggccgcat agataactga tccagtgtgc tggaattaat
     3661 tcgctgtctg cgagggccag ctgttggggt gagtactccc tctcaaaagc gggcatgact
     3721 tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac ctggcccgcg
     3781 gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa agacaatctt tttgttgtca
     3841 agcttgaggt gtggcaggct tgagatctgg ccatacactt gagtgacaat gacatccact
     3901 ttgcctttct ctccacaggt gtccactccc aggtccaact gcaggtcgag catgcatcta
     3961 gggcggccaa ttccgcccct ctcccccccc cccctctccc tccccccccc ctaacgttac
     4021 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     4081 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     4141 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     4201 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     4261 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     4321 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     4381 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     4441 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     4501 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     4561 ataagcttgc cacaacccgg gataattcct gcagccaata tgggatcggc cattgaacaa
     4621 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4681 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4741 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4801 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4861 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4921 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4981 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     5041 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     5101 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgatgatctc
     5161 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5221 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5281 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5341 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5401 tgaggggatc aattctctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     5461 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     5521 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5581 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5641 atcaatgtat cttaacgcgt cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5701 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5761 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5821 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5881 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5941 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6001 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6061 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6121 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6181 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6241 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6301 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6361 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6421 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6481 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6541 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6601 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6661 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6721 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6781 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6841 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6901 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6961 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7021 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7081 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7141 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7201 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7261 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7321 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7381 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7441 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7501 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7561 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7621 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7681 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7741 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7801 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7861 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
