LOCUS       sequence-318682-        7791 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 7791)
  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 7791)
  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: TRPML3-CFP
FEATURES             Location/Qualifiers
     source          1..7791
                     /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      707..716
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2378..2380,2381..3094)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     HFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2420..2441)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2681..2700)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3028..3049
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3110..3128)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3111..3128)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3148..3165)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3154..3378
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3424..3852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3511..3530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3721..3742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3861..3881)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3866..4195
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4046..4181
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4108..4127
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4262..5056
                     /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(4316..4335)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4926..4945
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5230..5351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5267..5286)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5321..5340
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5400..5416)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5400..5416)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5413..5435)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5424..5440
                     /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(5448..5454,5455..5472,5473..5478))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5493..5514
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5631..5648)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5802..6390)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5882..5901)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6561..7352,7353..7421))
                     /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     7184..7203
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7422..7526)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7601..7620)
                     /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 ggtaccgagc tcggatccac tagtccagtg tggtggaatt gcccttgcca ccatggcaaa
      721 tcccgaggtg ctggttagca gctgcagagc tcgccaagat gaaagcccct gcactttcca
      781 cccgagctcg tccccgtcag agcagcttct cttagaagac cagatgaggc ggaaactcaa
      841 gttctttttt atgaatcctt gtgagaagtt ctgggctcgg ggtaggaagc catggaaact
      901 tgccatacag attctgaaaa tcgcgatggt gactatccag ctggttctgt ttggactaag
      961 taaccagatg gtagtagctt tcaaagagga gaacactata gccttcaaac acctcttcct
     1021 aaagggctac atggatcgaa tggacgacac ctatgcagtg tacactcaga gtgaagtgta
     1081 tgaccagatc atctttgcag tgacccagta cttgcagctt cagaacatct ccgtgggcaa
     1141 tcacgcttat gagaacaagg ggactaagca gtcggcgatg gcaatctgtc agcacttcta
     1201 caggcgagga accatctgcc ccgggaacga cacctttgac atcgatccag aagttgaaac
     1261 agaatgtttc cttgtagagc cagatgaagc ttcccacctt ggaacgcctg gagaaaataa
     1321 actcaacctg agcctggact accacagact tctgacggtg gagctccagt ttaagctcaa
     1381 agccatcaat ctgcagacag ttcgacacca ggagcttcct gactgttacg actttacact
     1441 gactataaca ttcgacaaca aggctcacag tggaagaatc aaaataagct tagacaacga
     1501 catttctatc aaagaatgca aagactggca tgtgtctgga tcaattcaga agaacacaca
     1561 ctacatgatg atctttgatg cctttgtcat tctgacctgc ttggcctcac tggtgctgtg
     1621 tgccaggtct gtgattaggg gtcttcagct tcagcaggag tttgtcaact tcttccttct
     1681 tcactacaag aaggaagttt cggcctctga tcagatggag ttcatcaacg ggtggtacat
     1741 tatgatcatc attagtgaca tattgacaat cgttggatca gttctgaaaa tggaaatcca
     1801 agccaagagt ctcacaagct atgatgtctg cagcatactt ctcgggacgt caactatgct
     1861 cgtgtggctt ggagttatcc gatacctggg tttctttgcg aagtacaatc tccttattct
     1921 gaccctccag gcagcgctgc ccaacgtcat gaggttctgt tgctgcgctg ctatgatcta
     1981 tctaggctat tgcttttgcg gatggattgt gctgggccct taccatgaga agttccgttc
     2041 cctgaacagg gtctccgagt gcctgttctc gctgataaac ggagacgata tgttttccac
     2101 atttgcgaaa atgcagcaga agagttacct ggtgtggctg ttcagccgag tctacctgta
     2161 ctcgttcatc agcctcttca tttacatgat tctgagcctt ttcatcgcgc tcatcacaga
     2221 cacatacgaa acaattaagc actaccagca agatggcttc ccagagacgg aacttcgaaa
     2281 gtttatagcg gaatgcaaag acctccccaa ctccggaaaa tacagattag aagatgaccc
     2341 tccgggttct ttactctgct gctgcaaaaa gctcgaggtg agcaagggcg aggagctgtt
     2401 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaggttcag
     2461 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2521 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctggggcgt
     2581 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2641 gcccgaaggc tacgtccagg agcgtaccat cttcttcaag gacgacggca actacaagac
     2701 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2761 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acatcagcca
     2821 caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggcccact tcaagatccg
     2881 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2941 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     3001 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3061 gatcactctc ggcatggacg agctgtacaa gtaatctaga gggccctatt ctatagtgtc
     3121 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3181 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3241 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3301 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3361 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3421 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3481 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3541 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3601 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3661 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3721 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3781 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3841 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3901 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3961 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4021 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4081 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     4141 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4201 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     4261 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4321 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4381 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4441 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4501 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4561 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4621 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4681 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4741 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4801 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4861 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4921 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4981 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5041 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5101 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5161 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5221 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5281 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5341 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5401 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5461 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5521 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5581 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5641 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5701 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5761 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5821 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5881 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5941 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6001 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6061 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6121 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6181 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6241 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6301 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6361 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6421 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6481 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6541 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6601 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6661 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6721 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6781 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6841 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6901 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6961 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7021 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7081 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7141 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7201 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7261 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7321 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7381 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7441 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7501 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7561 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7621 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7681 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     7741 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt t
//
