LOCUS       sequence-460714-        6413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HRS 2xFYVE tagged with StayGold in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6413)
  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 6413)
  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: "Nat 
            Commun."; date: "2024-07-10"; volume: "15(1):5813. doi"; pages: " 
            10.1038/s41467-024-49876-9"
COMMENT     Sequence Label: pSems StayGold-2xFYVE
FEATURES             Location/Qualifiers
     source          1..6413
                     /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"
     intron          2204..2433
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2416..2435
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2491..3064
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2658..2675)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2885..2904
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3100..3903
                     /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(3163..3182)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3773..3792
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4033..4154
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4070..4089)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4256..4272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4256..4272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4269..4291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4280..4296
                     /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(4304..4310,4311..4328,4329..4334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4349..4370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4487..4504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4658..5246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4738..4757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5417..6208,6209..6277))
                     /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     6040..6059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6278..6382)
                     /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 gagctcggac cggtgctagc atcgatatcg aattcatggc cagcaccggg gaggagctgt
      961 tcaccggggt ggtgcccttc aagttccagc tgaagggcac catcaacggc aagagcttca
     1021 ccgtggaagg cgagggcgag ggcaatagcc acgagggcag ccacaaaggc aagtacgtgt
     1081 gcaccagcgg caaactgcca atgtcttggg ccgccctggg aactagcttc ggctatggca
     1141 tgaagtacta caccaagtac cccagcggcc tgaagaactg gttccacgag gtgatgcccg
     1201 agggcttcac ctacgacaga cacatccagt acaagggcga cggcagcatc cacgccaagc
     1261 accagcactt catgaagaac ggcacctacc acaacatcgt ggagttcacc ggccaggact
     1321 tcaaggagaa cagccccgtg ctgaccggcg acatgaacgt gagcctgccc aacgaggtgc
     1381 agcacatccc cagagatgac ggcgtggagt gcccagtgac cctgctgtac cctctgctga
     1441 gcgacaagag caagtgcgtg gaggcccacc agaacaccat ctgcaagccc ctgcacaatc
     1501 agccagcccc cgatgtgcca taccactgga tcagaaagca gtacacccag agcaaggacg
     1561 acaccgagga gagagaccac atctgccaga gcgagaccct ggaggcccac ctgctcgagc
     1621 tcaagcttcg aattgaattc gaaagtgatg ccatgttcgc tgctgaaaga gcccctgact
     1681 gggtggatgc tgaggaatgc catcggtgca gagtacagtt tggggtggtg acccgcaagc
     1741 atcactgccg agcatgtggg cagatcttct gtggcaagtg ctcctccaag tactccacca
     1801 tccccaagtt cggcattgag aaggaggtgc gcgtgtgtga gccctgctat gagcagctga
     1861 acaagaaggc acaaggacaa ggatccgaaa gtgatgccat gttcgctgct gaaagagccc
     1921 ctgactgggt ggatgctgag gaatgccatc ggtgcagagt acagtttggg gtggtgaccc
     1981 gcaagcatca ctgccgagca tgtgggcaga tcttctgtgg caagtgctcc tccaagtact
     2041 ccaccatccc caagttcggc attgagaagg aggtgcgcgt gtgtgagccc tgctatgagc
     2101 agctgaacaa gaaggcataa gtcgacggta ccgcggcggc cgcatagata actgatccag
     2161 tgtgctggaa ttaattcgct gtctgcgagg gccagctgtt ggggtgagta ctccctctca
     2221 aaagcgggca tgacttctgc gctaagattg tcagtttcca aaaacgagga ggatttgata
     2281 ttcacctggc ccgcggtgat gcctttgagg gtggccgcgt ccatctggtc agaaaagaca
     2341 atctttttgt tgtcaagctt gaggtgtggc aggcttgaga tctggccata cacttgagtg
     2401 acaatgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcagg
     2461 tcgagcatgc atctagggcg gccaattccg cccctctccc yccccccccc ctaacgttac
     2521 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     2581 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     2641 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     2701 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     2761 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     2821 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     2881 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     2941 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     3001 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     3061 ataagcttgc cacaacccgg gataattcct gcagccaata tgggatcggc cattgaacaa
     3121 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3181 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     3241 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     3301 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     3361 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3421 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3481 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3541 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3601 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgatgatctc
     3661 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3721 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3781 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3841 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3901 tgaggggatc aattctctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     3961 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     4021 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4081 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4141 atcaatgtat cttaacgcgt cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4201 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4321 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4441 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4501 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4561 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4621 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4681 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4741 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4801 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4861 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4921 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4981 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5041 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5101 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5161 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5221 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5281 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5341 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5401 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5461 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5521 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5581 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5641 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5701 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5761 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5821 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5881 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5941 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6001 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6061 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6121 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6181 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6241 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6301 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6361 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
