LOCUS       sequence-316886-        7704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7704)
  AUTHORS   Venkatachalam K, Hofmann T, Montell C
  TITLE     Lysosomal localization of TRPML3 depends on TRPML2 and the 
            mucolipidosis-associated protein TRPML1.
  JOURNAL   J Biol Chem. 2006 Jun 23. 281(25):17517-27.
  PUBMED    16606612
REFERENCE   2  (bases 1 to 7704)
  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: "J Biol 
            Chem. 2006 Jun 23. 281(25):17517-27."
COMMENT     Sequence Label: TRPML2-YFP
FEATURES             Location/Qualifiers
     source          1..7704
                     /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"
     regulatory      665..674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2291..2293,2294..3007)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2333..2354)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2594..2613)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2941..2962
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3023..3041)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3024..3041)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3061..3078)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3067..3291
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3337..3765
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3424..3443)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3634..3655
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3774..3794)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3779..4108
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3959..4094
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4021..4040
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4175..4969
                     /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(4229..4248)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4839..4858
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5143..5264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5180..5199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5234..5253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5313..5329)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5313..5329)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5326..5348)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5337..5353
                     /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(5361..5367,5368..5385,5386..5391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5406..5427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5544..5561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5715..6303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5795..5814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6474..7265,7266..7334))
                     /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     7097..7116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7335..7439)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7514..7533)
                     /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 acccaagctt
      661 accagccacc atggcgtctc tcgattctga ggtgagggaa gagtgtctga gggaagacct
      721 gaagttctac ttcatgagtc cctgtgagaa gtaccgagcc agacgtcaga tcccgtggaa
      781 actgggcttg cagatattga agatagtcat ggtaaccaca cagcttgttc ggtttggctt
      841 aagtaaccag ttggtggtgg ctttcaaaga agataacact gttgctttta agcacttgtt
      901 tttgaaagga ttttctggag ttgatgaaga tgactacagc tgtagcatat acactcagga
      961 gaacacctat gagagcatct tttttgctat aaagcagtat cgtcatctga agaatatttc
     1021 cctggcaacc cttggttatg gagaaagtga agacaataga actggcttga aggtctgtaa
     1081 gcagcactac aagacagggg ccatgttttc ttctaacgag accctgaaca ttgacagtga
     1141 cattgagaca gactgcattc acctggacct tcaggtgctc accacggaac ccgaggactg
     1201 ggcacagaca tctttcttca ggctggattt ttatcggctc gtgcaagttg atatctcctt
     1261 tgccctcaaa ggaattgacc tacaggcagt tcattcccga gagataccgg actgttacct
     1321 ctttcagaat acgattacct tcgacaacac agcccacagt gggaaaatca aaatctatct
     1381 caacagcgag gccaacatcg aagagtgtaa aaacatgaac atatctgggt ctactcagag
     1441 aagcactcac tacctcctgg tgtttgatgt gttcgtcatt atgatctgcc tggcatccct
     1501 tatcctgtgc acgagatcca ttgttcttgc tctgaggctg aggaagagat ttctaaactt
     1561 cttcctggag aagtacaaac agcgcgtgtg tggcgctgac cagtgggaat tcgtcaacgg
     1621 atggtacgtc ctggtcacta tcagcgacct catgacaata attggatcca tattgaagat
     1681 ggaaatcaaa gccaagaagc tcacaaacta tgacgtgtgc agcatcttgc tcgggacgtc
     1741 gacgctcttt gtctgggttg gagtcatcag atatctgggt tactttcaga cgtacaacgt
     1801 gctcatttta accatgcaag cctcactgcc caaagttctc cggttctgtg cttgcgctgg
     1861 catgatctac ctggggtaca ccttctgcgg ctggattgtc ttaggaccgt accacgagaa
     1921 gtttgaaaac ctgaacatag tcgctgaatg cctgttttct ctggtcaacg gcgatgacat
     1981 gtttgccacg ttcgctcaga tccagcagaa gagcattctg gtgtggctgt tcagccgcct
     2041 ctacctgtat tcctttatca gcctattcat ttacatggtc ctcagcctct tcatcgcgct
     2101 catcaccgac tcttaccaca ccattaagaa gtaccagcag catgggtttc ctgaaacaga
     2161 tctgcagaaa ttcctgaagg agagcggaag caaagatggg taccagaagc agccatcggc
     2221 cttgctgtcc tgcctctgct gtctgaggag gagaagaagc aatgaccact tgattctcat
     2281 tgacctcgag gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2341 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2401 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2461 gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct accccgacca
     2521 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2581 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2641 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2701 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2761 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2821 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2881 caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2941 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3001 caagtaatct agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag
     3061 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3121 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3181 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3241 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3301 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3361 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3421 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3481 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3541 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3601 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3661 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3721 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3781 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3841 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3901 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3961 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4021 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     4081 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     4141 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4201 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4261 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4321 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     4381 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4441 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4501 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4561 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4621 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4681 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4741 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4801 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4861 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4921 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4981 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5041 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5101 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5161 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5221 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5281 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5341 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5401 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5701 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5761 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5821 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5881 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5941 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6001 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6061 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6121 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6181 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6241 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6301 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6361 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6421 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6481 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6541 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6601 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6661 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6721 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6781 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6841 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6901 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6961 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7021 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7081 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7141 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7201 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7261 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7321 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7381 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7441 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     7501 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7561 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     7621 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     7681 atgtacgggc cagatatacg cgtt
//
