LOCUS       sequence-388907-        6859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RUFY1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6859)
  AUTHORS   Keren-Kaplan T, Saric A, Ghosh S, Williamson CD, Jia R, Li Y, 
            Bonifacino JS
  TITLE     RUFY3 and RUFY4 are ARL8 effectors that promote coupling of 
            endolysosomes to dynein-dynactin.
  JOURNAL   Nat Commun. 2022 Mar 21;13(1):1506. doi: 10.1038/s41467-022-28952-y.
  PUBMED    35314674
REFERENCE   2  (bases 1 to 6859)
  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: "2022-03-21"; volume: "13(1):1506. doi"; pages: " 
            10.1038/s41467-022-28952-y"
COMMENT     Sequence Label: RUFY1-GFP
FEATURES             Location/Qualifiers
     source          1..6859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(188..207)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    232..279
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      608..1196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1097..1116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1371..1674
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1675..1878
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1829..1849
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4109..4118
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4115..4117,4118..4120,4121..4834)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4160..4181)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4421..4440)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4768..4789
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4957..5078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4994..5013)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5048..5067
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5085..5540)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5222..5243)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5434..5453
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5567..5671
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5673..6030
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5673..5864
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5696..5716)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5881..6016
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6065..6859
                     /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             6065..6859
                     /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(6119..6138)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6729..6748
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
       61 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
      121 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
      181 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
      241 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
      301 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
      361 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
      421 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
      481 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
      541 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      601 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      661 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      721 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      781 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      841 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      901 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      961 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1021 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1081 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1141 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1201 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     1261 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa
     1321 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     1381 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     1441 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     1501 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     1561 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     1621 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     1681 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     1741 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     1801 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     1861 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggactcag
     1921 atctcgagct caagcttcga tggccgaccg ggaaggcggc tgcgctgctg ggcgggggcg
     1981 ggagctggag ccggagctgg agccggggcc ggggcccggg tcagcgcttg agccgggaga
     2041 agagtttgag atcgtggacc gaagccagct gcccggccca ggcgacctgc ggagcgcaac
     2101 gaggccgcgg gcggccgagg gctggtcggc gcccatcctg accctggcac gcagggccac
     2161 cgggaacctg tcggcgagct gcgggagcgc gctgcgcgcg gccgcggggc tgggcggcgg
     2221 ggacagcggg gacggcacgg cgcgcgcagc ttctaagtgc cagatgatgg aggagcgtgc
     2281 caacctgatg cacatgatga aactcagcat caaggtgttg ctccagtcgg ctctgagcct
     2341 gggccgcagc ctggatgcgg accatgcccc cttgcagcag ttctttgtag tgatggagca
     2401 ctgcctcaaa catgggctga aagttaagaa gagttttatt ggccaaaata aatcattctt
     2461 tggtcctttg gagctggtgg agaaactttg tccagaagca tcagatatag cgactagtgt
     2521 cagaaatctt ccagaattaa agacagctgt gggaagaggc cgagcgtggc tttatcttgc
     2581 actcatgcaa aagaaactgg cagattatct gaaagtgctt atagacaata aacatctctt
     2641 aagcgagttc tatgagcctg aggctttaat gatggaggaa gaagggatgg tgattgttgg
     2701 tctgctggtg ggactcaatg ttctcgatgc caatctctgc ttgaaaggag aagacttgga
     2761 ttctcaggtt ggagtaatag atttttccct ctaccttaag gatgtgcagg atcttgatgg
     2821 tggcaaggag catgaaagaa ttactgatgt ccttgatcaa aaaaattatg tggaagaact
     2881 taaccggcac ttgagctgca cagttgggga tcttcaaacc aagatagatg gcttggaaaa
     2941 gactaactca aagcttcaag aagagctttc agctgcaaca gaccgaattt gctcacttca
     3001 agaagaacag cagcagttaa gagaacaaaa tgaattaatt cgagaaagaa gtgaaaagag
     3061 tgtagagata acaaaacagg ataccaaagt tgagctggag acttacaagc aaactcggca
     3121 aggtctggat gaaatgtaca gtgatgtgtg gaagcagcta aaagaggaga agaaagtccg
     3181 gttggaactg gaaaaagaac tggagttaca aattggaatg aaaaccgaaa tggaaattgc
     3241 aatgaagtta ctggaaaagg acacccacga gaagcaggac acactagttg ccctccgcca
     3301 gcagctggaa gaagtcaaag cgattaattt acagatgttt cacaaagctc agaatgcaga
     3361 gagcagtttg cagcagaaga atgaagccat cacatccttt gaaggaaaaa ccaaccaagt
     3421 tatgtccagc atgaaacaaa tggaagaaag gttgcagcac tcggagcggg cgaggcaggg
     3481 ggctgaggag cggagccaca agctgcagca ggagctgggc gggaggatcg gcgccctgca
     3541 gctgcagctc tcccagctgc acgagcaatg ctcaagcctg gagaaagaat tgaaatcaga
     3601 aaaagagcaa agacaggctc ttcagcgcga attacagcac gagaaagaca cttcctctct
     3661 actcaggatg gagctgcaac aagtggaagg actgaaaaag gagttgcggg agcttcagga
     3721 cgagaaggca gagctgcaga agatctgcga ggagcaggaa caagccctcc aggaaatggg
     3781 cctgcacctc agccagtcca agctgaagat ggaagatata aaagaagtga accaggcact
     3841 gaagggccac gcctggctga aagatgacga agcgacacac tgtaggcagt gtgagaagga
     3901 gttctccatt tcccggagaa agcaccactg ccggaactgt ggccacatct tctgcaacac
     3961 ctgctccagc aacgagctgg ccctgccctc ctaccccaag ccggtgcgag tgtgcgacag
     4021 ctgccacacc ctgctcctgc agcgctgctc ctccacggcc tccaaaattc tgcagtcgac
     4081 ggtaccgcgg gcccgggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt
     4141 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     4201 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     4261 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     4321 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4381 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     4441 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     4501 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     4561 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     4621 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     4681 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag
     4741 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     4801 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc
     4861 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     4921 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     4981 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5041 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     5101 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5161 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5221 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5281 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5341 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5401 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5461 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     5521 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5581 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5641 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     5701 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5761 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     5821 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5881 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5941 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     6001 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     6061 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     6121 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     6181 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     6241 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     6301 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     6361 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     6421 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     6481 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     6541 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     6601 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     6661 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     6721 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     6781 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     6841 tcttgacgag ttcttctga
//
