LOCUS       sequence-488270-        6413 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Expresses TRPML1 mutant with C-terminus mScarlet-I tag in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6413)
  TITLE     Martin lab plasmids
REFERENCE   2  (bases 1 to 6413)
  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 V432P mScarlet-I
FEATURES             Location/Qualifiers
     source          1..6413
                     /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      2314..2323
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2320..3015
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2453..2471)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2721..2740
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3141..3262
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3178..3197)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3232..3251
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3269..3724)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3406..3427)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3618..3637
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3751..3855
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3857..4214
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3857..4048
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3880..3900)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4065..4200
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4249..5043
                     /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             4249..5043
                     /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(4303..4322)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4913..4932
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5231..5250)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5275..5322
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5651..6239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6140..6159
                     /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 ccggactcag atctcgagat gacagcccca gcaggtccac gcggctcaga gaccgagcgg
      601 cttctgaccc ccaaccccgg gtatgggacc caggcggggc cttcaccggc ccctccgaca
      661 cccccagaag aggaagacct tcgccgtcgt ctcaaatact ttttcatgag tccctgcgac
      721 aagtttcgag ccaagggccg caagccctgc aagctgatgc tgcaagtggt caagatcctg
      781 gtggtcacgg tgcagctcat cctgtttggg ctcagtaatc agctggctgt gacattccgg
      841 gaagagaaca ccatcgcctt ccgacacctc ttcctgctgg gctactcgga cggagcggat
      901 gacaccttcg cagcctacac gcgggagcag ctgtaccagg ccatcttcca tgctgtggac
      961 cagtacctgg cgttgcctga cgtgtcactg ggccggtatg cgtatgtccg tggtgggggt
     1021 gacccttgga ccaatggctc agggcttgct ctctgccagc ggtactacca ccgaggccac
     1081 gtggacccgg ccaacgacac atttgacatt gatccgatgg tggttactga ctgcatccag
     1141 gtggatcccc ccgagcggcc ccctccgccc cccagcgacg atctcaccct cttggaaagc
     1201 agctccagtt acaagaacct cacgctcaaa ttccacaagc tggtcaatgt caccatccac
     1261 ttccggctga agaccattaa cctccagagc ctcatcaata atgagatccc ggactgctat
     1321 accttcagcg tcctgatcac gtttgacaac aaagcacaca gtgggcggat ccccatcagc
     1381 ctggagaccc aggcccacat ccaggagtgt aagcacccca gtgtcttcca gcacggagac
     1441 aacagcttcc ggctcctgtt tgacgtggtg gtcatcctca cctgctccct gtccttcctc
     1501 ctctgcgccc gctcactcct tcgaggcttc ctgctgcaga acgagtttgt ggggttcatg
     1561 tggcggcagc ggggacgggt catcagcctg tgggagcggc tggaatttgt caatggctgg
     1621 tacatcctgc tcgtcaccag cgatgtgctc accatctcgg gcaccatcat gaagatcggc
     1681 atcgaggcca agaacttggc gagctacgac gtctgcagca tcctcctggg cacctcgacg
     1741 ctgctggtgt gggtgggcgt gatccgctac ctgaccttct tccacaacta caatatcctc
     1801 atcgccacac tgcgggtggc cctgcccagc gtcatgcgct tctgctgctg ccccgctgtc
     1861 atctacctgg gctactgctt ctgtggctgg atcgtgctgg ggccctatca tgtgaagttc
     1921 cgctcactct ccatggtgtc tgagtgcctg ttctcgctca tcaatgggga cgacatgttt
     1981 gtgacgttcg ccgccatgca ggcgcagcag ggccgcagca gcctggtgtg gctcttctcc
     2041 cagctctacc tttactcctt catcagcctc ttcatctaca tggtgctcag cctcttcatc
     2101 gcgctcatca ccggcgccta cgacaccatc aagcatcccg gcggcgcagg cgcagaggag
     2161 agcgagctgc aggcctacat cgcacagtgc caggacagcc ccacctccgg caagttccgc
     2221 cgcgggagcg gctcggcctg cagccttctc tgctgctgcg gaagggaccc ctcggaggag
     2281 cattcgctgc tggtgaatcc ggatccaccg gtcgccacca tggtgagcaa gggcgaggca
     2341 gtgatcaagg agttcatgcg gttcaaggtg cacatggagg gctccatgaa cggccacgag
     2401 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     2461 aaggtgacca agggtggccc cctgcccttc tcctgggaca tcctgtcccc tcagttcatg
     2521 tacggctcca gggccttcat caagcacccc gccgacatcc ccgactacta taagcagtcc
     2581 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc
     2641 gtgacccagg acacctccct ggaggacggc accctgatct acaaggtgaa gctccgcggc
     2701 accaacttcc ctcctgacgg ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc
     2761 accgagcggt tgtaccccga ggacggcgtg ctgaagggcg acattaagat ggccctgcgc
     2821 ctgaaggacg gcggccgcta cctggcggac ttcaagacca cctacaaggc caagaagccc
     2881 gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg acatcacctc ccacaacgag
     2941 gactacaccg tggtggaaca gtacgaacgc tccgagggcc gccactccac cggcggcatg
     3001 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg
     3061 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     3121 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     3181 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3241 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     3301 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     3361 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     3421 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3481 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     3541 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     3601 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3661 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3721 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3781 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3841 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     3901 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3961 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4021 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4081 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4141 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4201 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4261 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4321 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4381 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     4441 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4501 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4561 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4621 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4681 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4741 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     4801 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4861 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4921 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4981 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5041 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5101 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5161 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     5221 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     5281 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     5341 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     5401 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     5461 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     5521 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5581 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5641 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5701 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5761 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5821 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5881 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5941 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6001 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6061 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6121 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6181 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6241 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6301 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
     6361 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccg
//
