LOCUS       sequence-488276-        6463 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Expresses TRPML1 mutant with C-terminus EGFP tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6463)
  TITLE     Martin lab plasmids
REFERENCE   2  (bases 1 to 6463)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: TRPML1 Y499A-EGFP
FEATURES             Location/Qualifiers
     source          1..6463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(553..555,556..558,559..1269)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(553..555,556..558,559..1269)
                     /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(598..619)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(859..878)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1206..1227
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3191..3312
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3228..3247)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3282..3301
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3319..3774)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3456..3477)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3668..3687
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3801..3905
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3907..4264
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3907..4098
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3930..3950)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4115..4250
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4299..5093
                     /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             4299..5093
                     /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(4353..4372)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4963..4982
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5281..5300)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5325..5372
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5701..6289
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6190..6209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      601 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1201 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1261 ctgtacaagt ccggactcag atctcgagca acagcaccag caggtccacg cggctcagag
     1321 accgagcggc ttctgacccc caaccccggg tatgggaccc aggcggggcc ttcaccggcc
     1381 cctccgacac ccccagaaga ggaagacctt cgccgtcgtc tcaaatactt tttcatgagt
     1441 ccctgcgaca agtttcgagc caagggccgc aagccctgca agctgatgct gcaagtggtc
     1501 aagatcctgg tggtcacggt gcagctcatc ctgtttgggc tcagtaatca gctggctgtg
     1561 acattccggg aagagaacac catcgccttc cgacacctct tcctgctggg ctactcggac
     1621 ggagcggatg acaccttcgc agcctacacg cgggagcagc tgtaccaggc catcttccat
     1681 gctgtggacc agtacctggc gttgcctgac gtgtcactgg gccggtatgc gtatgtccgt
     1741 ggtgggggtg acccttggac caatggctca gggcttgctc tctgccagcg gtactaccac
     1801 cgaggccacg tggacccggc caacgacaca tttgacattg atccgatggt ggttactgac
     1861 tgcatccagg tggatccccc cgagcggccc cctccgcccc ccagcgacga tctcaccctc
     1921 ttggaaagca gctccagtta caagaacctc acgctcaaat tccacaagct ggtcaatgtc
     1981 accatccact tccggctgaa gaccattaac ctccagagcc tcatcaataa tgagatcccg
     2041 gactgctata ccttcagcgt cctgatcacg tttgacaaca aagcacacag tgggcggatc
     2101 cccatcagcc tggagaccca ggcccacatc caggagtgta agcaccccag tgtcttccag
     2161 cacggagaca acagcttccg gctcctgttt gacgtggtgg tcatcctcac ctgctccctg
     2221 tccttcctcc tctgcgcccg ctcactcctt cgaggcttcc tgctgcagaa cgagtttgtg
     2281 gggttcatgt ggcggcagcg gggacgggtc atcagcctgt gggagcggct ggaatttgtc
     2341 aatggctggt acatcctgct cgtcaccagc gatgtgctca ccatctcggg caccatcatg
     2401 aagatcggca tcgaggccaa gaacttggcg agctacgacg tctgcagcat cctcctgggc
     2461 acctcgacgc tgctggtgtg ggtgggcgtg atccgctacc tgaccttctt ccacaactac
     2521 aatatcctca tcgccacact gcgggtggcc ctgcccagcg tcatgcgctt ctgctgctgc
     2581 gtggctgtca tctacctggg ctactgcttc tgtggctgga tcgtgctggg gccctatcat
     2641 gtgaagttcc gctcactctc catggtgtct gagtgcctgt tctcgctcat caatggggac
     2701 gacatgtttg tgacgttcgc cgccatgcag gcgcagcagg gccgcagcag cctggtgtgg
     2761 ctcttctccc agctctacct tgcctccttc atcagcctct tcatctacat ggtgctcagc
     2821 ctcttcatcg cgctcatcac cggcgcctac gacaccatca agcatcccgg cggcgcaggc
     2881 gcagaggaga gcgagctgca ggcctacatc gcacagtgcc aggacagccc cacctccggc
     2941 aagttccgcc gcgggagcgg ctcggcctgc agccttctct gctgctgcgg aagggacccc
     3001 tcggaggagc attcgctgct ggtgaattga gaattctgca gtcgacggta ccgcgggccc
     3061 gggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     3121 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3181 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3241 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3301 caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3361 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3421 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3481 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3541 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3601 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3661 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3721 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3781 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3841 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3901 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3961 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4021 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4081 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4141 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4201 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4261 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     4321 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4381 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4441 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4501 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4561 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4621 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4681 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4741 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4801 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4861 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4921 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4981 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     5041 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     5101 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     5161 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5221 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     5281 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     5341 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5401 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5461 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5521 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5581 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5641 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5701 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5761 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5821 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5881 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5941 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6001 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6061 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6121 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6181 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6241 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6301 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6361 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     6421 caattacggg gtcattagtt catagcccat atatggagtt ccg
//
