LOCUS       sequence-272059-        7367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding outer inner membrane localization of SNAP-Tag and 
            EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7367)
  AUTHORS   Farrants H, Tarnawski M, Muller TG, Otsuka S, Hiblot J, Koch B, 
            Kueblbeck M, Krausslich HG, Ellenberg J, Johnsson K
  TITLE     Chemogenetic Control of Nanobodies.
  JOURNAL   Nat Methods. 2020 Feb 17. pii: 10.1038/s41592-020-0746-7. doi: 
            10.1038/s41592-020-0746-7.
  PUBMED    32066961
REFERENCE   2  (bases 1 to 7367)
  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 
            Methods. 2020 Feb 17. pii: 10.1038/s41592-020-0746-7. doi: 
            10.1038/s41592-020-0746-7."
COMMENT     Sequence Label: pCDNA3.0_LynSNAP IRES EGFP
FEATURES             Location/Qualifiers
     source          1..7367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1285..1309)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1338..1904
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1355..1906
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1499..1516)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1726..1745
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1909..1911,1912..1914,1915..2625)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1909..1911,1912..1914,1915..2625)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1954..1975)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2215..2234)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2562..2583
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2686..2704)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2687..2704)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2724..2741)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2730..2954
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3000..3428
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3087..3106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3297..3318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3437..3457)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3442..3771
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3622..3757
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3684..3703
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3838..4632
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3892..3911)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4502..4521
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4806..4927
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4843..4862)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4897..4916
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4976..4992)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4976..4992)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4989..5011)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5000..5016
                     /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(5024..5030,5031..5048,5049..5054))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5069..5090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5207..5224)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5378..5966)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5458..5477)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6137..6928,6929..6997))
                     /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     6760..6779
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6998..7102)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7177..7196)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccagcggc
      661 cgcaccatgg gatgtataaa atcaaaaggg aaagacagcg cgggagcaga tagtgctggt
      721 agtgctggtg ataaagactg cgaaatgaaa cgcactaccc tggacagccc gctgggtaaa
      781 ctggaactga gcggttgcga gcagggcctg cacgaaatca ttttcctggg caaaggtacg
      841 agcgctgcgg atgcggtcga agtaccggct ccggctgcgg ttctgggtgg cccggagccg
      901 ctgatgcagg ctaccgcatg gctgaacgca tattttcacc agccagaggc gattgaagag
      961 ttccctgtac cggccctgca tcacccggta tttcagcagg aatcctttac ccgtcaggta
     1021 ctgtggaaac tgctgaaagt agtgaaattc ggcgaggtga tctcctattc ccatctggcg
     1081 gcgctggctg gcaacccagc ggcaactgcg gcggtgaaaa ccgctctgtc tggtaatccg
     1141 gtgccaattc tgatcccgtg ccaccgtgtg gttcagggcg acctggacgt aggtggctac
     1201 gaaggcggcc tggctgtgaa agaatggctg ctggcgcacg aaggtcaccg tctgggtaaa
     1261 ccaggcctgg gctagtaagg atccacccag ctttcttgta caaagtggtg atcgcgtaaa
     1321 ctacgggctg caggaattcc gccccccccc cctaacgtta ctggccgaag ccgcttggaa
     1381 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     1441 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     1501 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     1561 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     1621 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     1681 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     1741 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     1801 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc
     1861 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataataccat ggtgagcaag
     1921 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1981 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2041 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2101 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2161 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2221 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2281 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2341 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2401 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2461 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2521 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2581 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg actcagatct
     2641 cgactagcta gtagctaact cgagcatgca tctagagggc cctattctat agtgtcacct
     2701 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2761 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2821 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2881 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2941 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3001 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3061 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3121 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3181 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3241 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3301 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3361 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3421 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3481 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3541 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3601 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3661 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3721 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3781 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3841 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3901 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3961 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4021 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4081 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4141 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4201 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4261 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4321 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4381 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4441 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4501 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4561 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4621 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4681 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4741 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4801 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4861 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4921 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4981 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5041 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5101 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5161 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5221 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5281 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5341 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5401 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5461 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5521 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5581 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5641 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5701 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5761 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5821 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5881 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5941 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6001 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6061 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6121 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6181 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6241 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6301 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6361 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6421 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6481 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6541 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6601 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6661 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6721 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6781 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6841 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6901 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6961 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7021 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7081 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7141 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     7201 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     7261 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     7321 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
