LOCUS       sequence-454826-        7145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse TMEM63A-W472K-eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7145)
  AUTHORS   Lowry AJ, Liang P, Song M, Serena Wan YC, Pei ZM, Yang H, Zhang Y
  TITLE     TMEM16 and OSCA/TMEM63 proteins share a conserved potential to 
            permeate ions and phospholipids.
  JOURNAL   bioRxiv [Preprint]. 2024 Sep 3:2024.02.04.578431. doi: 
            10.1101/2024.02.04.578431.
  PUBMED    38370744
REFERENCE   2  (bases 1 to 7145)
  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: "bioRxiv 
            [Preprint]. 2024 Sep 3:2024.02.04.578431. doi: 
            10.1101/2024.02.04.578431."
COMMENT     Sequence Label: TMEM63A-W472K-peGFP-N1
FEATURES             Location/Qualifiers
     source          1..7145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     CDS             join(3091..3093,3094..3096,3097..3810)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3091..3093,3094..3096,3097..3807)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3136..3157)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3397..3416)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3744..3765
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3933..4054
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3970..3989)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4024..4043
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4061..4516)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4198..4219)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4410..4429
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4543..4647
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4649..5006
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4649..4840
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4672..4692)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4857..4992
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5041..5835
                     /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             5041..5835
                     /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(5095..5114)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5705..5724
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6023..6042)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6067..6114
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6443..7031
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6932..6951
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgtccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat gaccagctcc ccgttcctgg atccctggcc gtccaaggct
      721 gtgtttgtcc gtgagcgcct gggtcttggc gaacgtccca atgactccta ttgctacaac
      781 tcagccaaaa acagcactgt gctccaaggg gtcacctttg gaggcatccc cactgtcctg
      841 ctcttagatg tcagttgctt tctgtttcta atcctggtgt tttccatcat aaggagaaga
      901 ttctgggatt atggccgcat agccctggta tcagaagcag gcagtgaggc caggttccag
      961 agactgtcct cttcctcctc agggcaacaa gactttgaaa acgagctggg atgctgtcct
     1021 tggctgacgg ccatcttccg cctgcatgac gaccagatcc tggagtggtg cggagaggac
     1081 gccatccact acctgtcttt tcagaggcac atcatcttcc tgctggtggt gatcagcttc
     1141 ttgtccctgt gtgtcatctt gcctgtcaac ctctcgggag acttgctggg taaagaccct
     1201 tacagctttg ggaggacaac gatagctaac ctgcagactg acaatgacct cctctggctg
     1261 cacactgtct tcagcgtcat ctacctcttc ctcacggtgg gcttcatgtg gcatcacaca
     1321 cggtccatca gatacaaaga ggagagtctg gtgagacaaa ccttgttcat cacaggactc
     1381 cccagagaag ccagaaagga gactgtggag agccacttcc gagatgcgta tcccacgtgt
     1441 gaggtggtgg atgtccagct gtgctacagc gtggctaagc tgatctacct gtgcaaagag
     1501 agaaagaaga ccgagaagag ccttacctat tacacaaacc tgcaggcgaa gacaggccga
     1561 cggaccctca tcaaccccaa gccttgtggc cagttctgct gctgtgaagt tcagggttgt
     1621 gagcgggaag atgctatttc ttactacact cgaatgaatg acagcctgct ggagaggatc
     1681 acagccgaag agagccgggt ccaggaccag cccctgggaa tggcctttgt caccttccgt
     1741 gagaagtcca tggctaccta catactgaaa gacttcaatg cctgcaagtg ccagggcctt
     1801 cggtgcaaag gggagcccca gccatcctcc tatagcagag agctctgtgt ctccaagtgg
     1861 acggtcactt ttgcctccta tcctgaggac atctgctgga agaacctctc catccagggc
     1921 gttcgctggt ggctccagtg gctgggtatc aacttctctc tgtttgtggt gctgtttttc
     1981 ctgaccacgc cctccatcat tatgtctacc atggacaagt tcaatgttac caaacccatc
     2041 cacgcactga ataaccccgt catcagccag ttcttcccca cactcctgct gaagtccttc
     2101 tctgcgctgc ttccgtccat tgtctactac tccacgctgc tggagtccca ctggacaagg
     2161 tcaggggaaa accggatcat ggtgtcgaag gtctacatat ttttgatctt catggtgctg
     2221 atcctgccct ccctcggtct caccagcttg gatttcttct tccggtggct ctttgacaaa
     2281 acttcctcgg aaacctcaat caggctagag tgtgtcttcc tgccagacca gggtgccttc
     2341 tttgtgaact acgtcatcgc ctcagccttc atcggcagtg gcatggagct gttgcgcctg
     2401 cccggcctca tcctctatac cttccgcatg atcatggcca agacggccgc tgaccgcagg
     2461 aatgttaagc agaaccaggc ttttgaatac gagtttggag ccatgtatgc gtggatgctg
     2521 tgtgtcttca ccgtcatcat ggcttacagc atcacctgcc ccattatcgt gccctttggc
     2581 ctcatctaca tcctgcttaa gcacatggtg gaccggcata acctctactt cgcttacctc
     2641 ccgactaaac tggagaagag gatccacttc gccgccgtga accaggccct ggccgccccc
     2701 atcctctgcc tcttctggct cttctttttc tccttcctcc gtctgggtct gacggccccg
     2761 gccacgctct tcaccttcct ggttgttctg ctcaccatcc tggcctgcct gctgtacacc
     2821 tgctttggat gcttcaagca cctcagccca tggaactaca agacagaaga atcagctagt
     2881 gacaaaggaa gcgaagccga ggcccacgtg cccccgccat tcacacccta tgtgcctcgg
     2941 attctgaacg gtttggcctc agagaggaca gcgttatccc cacagcagca gcagacctac
     3001 ggtgccatcc gtaacatcag tgggactctt cctgggcagc ccgtggctca agatcctagt
     3061 ggcactgcgg cttatgccta ccaggaatcc atggtgagca agggcgagga gctgttcacc
     3121 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     3181 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3241 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     3301 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3361 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3421 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3481 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     3541 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     3601 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3661 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3721 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3781 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     3841 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3901 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3961 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4021 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     4081 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4141 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4201 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4261 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4321 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4381 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4441 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4501 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4561 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4621 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     4681 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4741 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4801 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4861 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4921 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4981 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     5041 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     5101 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     5161 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     5221 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     5281 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     5341 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     5401 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     5461 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5521 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     5581 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5641 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5701 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5761 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5821 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5881 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5941 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     6001 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     6061 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     6121 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     6181 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     6241 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     6301 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6361 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6421 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6481 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6541 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6601 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6661 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6721 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6781 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6841 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6901 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6961 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7021 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7081 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     7141 tgcat
//
