LOCUS       sequence-321989-        5021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in mammalian cells of GFP binding protein (GBP) tagged 
            with photoconvertable protein mEos2 to track GFP proteins of 
            interest to perform Fluorescent intrabody Localization Microscopy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5021)
  AUTHORS   Gormal RS, Padmanabhan P, Kasula R, Bademosi AT, Coakley S, 
            Giacomotto J, Blum A, Joensuu M, Wallis TP, Lo HP, Budnar S, Rae J, 
            Ferguson C, Bastiani M, Thomas WG, Pardon E, Steyaert J, Yap AS, 
            Goodhill GJ, Hilliard MA, Parton RG, Meunier FA
  TITLE     Modular transient nanoclustering of activated beta2-adrenergic 
            receptors revealed by single-molecule tracking of 
            conformation-specific nanobodies.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Nov 19. pii: 2007443117. doi: 
            10.1073/pnas.2007443117.
  PUBMED    33214152
REFERENCE   2  (bases 1 to 5021)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Nov 19. pii: 2007443117. doi: 
            10.1073/pnas.2007443117."
COMMENT     Sequence Label: pmEos2-N1 GBP (GBP-mEos2)
FEATURES             Location/Qualifiers
     source          1..5021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             264..605
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVSS"
     CDS             627..1307
                     /codon_start=1
                     /product="monomeric variant of green-to-red photoswitchable
                     fluorescent protein EosFP (McKinney et al., 2009)"
                     /label=mEos2
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICIAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIHMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGYHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     polyA_signal    1445..1566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1482..1501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1536..1555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1573..2028)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1710..1731)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1922..1941
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2055..2159
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2161..2518
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2161..2352
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2184..2204)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2369..2504
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2553..3347
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2553..3347
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2607..2626)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3217..3236
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3535..3554)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3579..3626
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3955..4543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4444..4463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4718..5021
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgagctcatg caggttcaac tggtggaaag cggcggtgct ctggtacaac
      301 cgggcggtag tctgcgcctg agctgtgccg caagcggttt cccagtcaac cgctactcta
      361 tgcgttggta tcgccaggcg cctggtaaag aacgtgaatg ggttgccggc atgagcagtg
      421 cgggcgatcg ttctagttac gaggactctg ttaaaggtcg ttttacaatt agccgtgatg
      481 atgcgcgcaa taccgtgtat ctgcaaatga acagtctgaa gccggaggac accgcagtat
      541 attattgcaa tgtcaacgtg gggtttgaat attggggcca ggggactcag gtgacggtga
      601 gctctaaaga tccaccggtc gccaccatga gtgcgattaa gccagacatg aagatcaaac
      661 tccgtatgga aggcaacgta aacgggcacc actttgtgat cgacggagat ggtacaggca
      721 agccttttga gggaaaacag agtatggatc ttgaagtcaa agagggcgga cctctgcctt
      781 ttgcctttga tatcctgacc actgcattcc attacggcaa cagggtattc gccaaatatc
      841 cagacaacat acaagactat tttaagcagt cgtttcctaa ggggtattcg tgggaacgaa
      901 gcttgacttt cgaagacggg ggcatttgca ttgccagaaa cgacataaca atggaagggg
      961 acactttcta taataaagtt cgattttatg gtaccaactt tcccgccaat ggtccagtta
     1021 tgcagaagaa gacgctgaaa tgggagccct ccactgagaa aatgtatgtg cgtgatggag
     1081 tgctgacggg tgatattcat atggctttgt tgcttgaagg aaatgcccat taccgatgtg
     1141 acttcagaac tacttacaaa gctaaggaga agggtgtcaa gttaccaggc taccactttg
     1201 tggaccactg cattgagatt ttaagccatg acaaagatta caacaaggtt aagctgtatg
     1261 agcatgctgt tgctcattct ggattgcctg acaatgccag acgataagag ctgtacaagt
     1321 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1381 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1441 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1501 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1561 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     1621 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     1681 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     1741 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     1801 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     1861 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     1921 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     1981 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2041 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2101 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     2161 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2221 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2281 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2341 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2401 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2461 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     2521 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     2581 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2641 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2701 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     2761 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     2821 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     2881 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     2941 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3001 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3061 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3121 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3181 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3241 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3301 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3361 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3421 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3481 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     3541 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     3601 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     3661 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     3721 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     3781 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     3841 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3901 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3961 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4021 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4081 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4141 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4201 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4261 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4321 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4381 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4441 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4501 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4561 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4621 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
     4681 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     4741 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     4801 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     4861 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     4921 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     4981 cttggcagta catctacgta ttagtcatcg ctattaccat g
//
