LOCUS       sequence-324225-        6720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MUTED in mammalian cells with a GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6720)
  AUTHORS   Bonet-Ponce L, Beilina A, Williamson CD, Lindberg E, Kluss JH, 
            Saez-Atienzar S, Landeck N, Kumaran R, Mamais A, Bleck CKE, Li Y, 
            Cookson MR
  TITLE     LRRK2 mediates tubulation and vesicle sorting from lysosomes.
  JOURNAL   Sci Adv. 2020 Nov 11;6(46). pii: 6/46/eabb2454. doi: 
            10.1126/sciadv.abb2454. Print 2020 Nov.
  PUBMED    33177079
REFERENCE   2  (bases 1 to 6720)
  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: "Sci Adv."; 
            date: "2020-11-11"; volume: "6(46). pii: 6/46/eabb2454. doi"; pages:
            " 10.1126/sciadv.abb2454. Print 2020 Nov"
COMMENT     Sequence Label: GFP-MUTED
FEATURES             Location/Qualifiers
     source          1..6720
                     /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"
     CDS             join(905..907,908..910,911..1621)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(938..966)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1561..1581
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    1643..1667
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2255..2279)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2311..2328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2317..2541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2587..3015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2674..2693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2884..2905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3024..3044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3029..3358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3209..3344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3271..3290
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3425..4219
                     /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(3479..3498)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4089..4108
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4393..4514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4430..4449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4484..4503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4563..4579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4563..4579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4576..4598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4587..4603
                     /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(4611..4617,4618..4635,4636..4641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4656..4677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4794..4811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4965..5553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5045..5064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5724..6515,6516..6584))
                     /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     6347..6366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6585..6689)
                     /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 gctggctaga
      901 caccatggcc agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      961 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgctac
     1021 atacggaaag cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc
     1081 aacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat
     1141 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat
     1201 atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac
     1261 ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg
     1321 acacaaactc gagtacaact ataactcaca caatgtatac atcacggcag acaaacaaaa
     1381 gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatggat ccgttcaact
     1441 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
     1501 ccattacctg tcgacacaat ctgccctttc gaaagatccc aacgaaaagc gtgaccacat
     1561 ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa
     1621 aagcggttcc ggaccggatc aaacaagttt gtacaaaaaa gcaggctccg aattcgccct
     1681 tgtggcggag ggacagagac ccctgtgggt tgtgaggccg ccccgggcgg tggcagcaag
     1741 aagagggact ccctggggac tgcgggctca gcgcacctca ttatcaagga tcttggagaa
     1801 attcattcaa ggcttttgga tcacagacca gttattcaag gtgaaactcg ttattttgta
     1861 aaagaatttg aagaaaaacg tggtcttcga gaaatgcgag ttcttgaaaa tttgaagaac
     1921 atgatccatg aaacaaatga acatactctt cccaaatgta gagacacaat gcgggacagc
     1981 ctcagccagg ttctccagag attgcaagca gctaatgact cagtctgtag actccaacag
     2041 agggaacagg aacgaaaaaa gattcatagt gaccacttag tagctagtga gaaacagcat
     2101 atgctccagt gggacaactt catgaaggag caacccaaca aaagggctga agtggatgaa
     2161 gagcacagaa aagccatgga aaggcttaaa gaacaatatg ctgagatgga gaaggaccta
     2221 gcgaaatttt caacctttta aaagggcgaa ttcgacccag ctttcttgta caaagtggtg
     2281 ataattaatt aagataaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     2341 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2401 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2461 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2521 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2581 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2641 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2701 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2761 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2821 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2881 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2941 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3001 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3121 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3181 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3241 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3301 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3361 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3421 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3481 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3541 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3601 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3661 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3721 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3781 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3841 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3901 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3961 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4021 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4081 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4141 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4201 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4261 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4321 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4381 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4441 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4501 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     4561 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4621 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4681 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4741 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4801 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4861 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4921 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4981 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5041 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5101 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5161 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5221 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5281 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5341 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5401 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5461 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5521 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5581 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5641 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5701 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5761 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5821 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5881 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5941 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6001 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6061 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6121 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6181 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6241 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6301 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6361 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6421 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6481 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6541 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6601 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6661 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
