LOCUS       sequence-454373-        7046 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses OSCA1.2-K435L-L438K-eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7046)
  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 7046)
  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: OSCA1.2-K435L-L438K-peGFP-N1
FEATURES             Location/Qualifiers
     source          1..7046
                     /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"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             1796..1804
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             join(2992..2994,2995..2997,2998..3711)
                     /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(2992..2994,2995..2997,2998..3708)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3037..3058)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3298..3317)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3645..3666
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3834..3955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3871..3890)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3925..3944
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3962..4417)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4099..4120)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4311..4330
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4444..4548
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4550..4907
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4550..4741
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4573..4593)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4758..4893
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4942..5736
                     /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             4942..5736
                     /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(4996..5015)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5606..5625
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5924..5943)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5968..6015
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6344..6932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6833..6852
                     /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 atgcccagta
      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 ggcgacactt caggatattg gtgtatcagc tgggataaac
      721 attctcagtg cctttgtttt cttcataatc tttgcggttt tgaggcttca gcctttcaat
      781 gatagagttt acttctcaaa atggtatctc aaggggttaa gaagcagccc tgctcgtggt
      841 ggcgcctttg cgcagaggtt tgtgaacctg gacttcaggt cttatatgaa gttcttgaat
      901 tggatgccag aggctctgaa gatgcctgag cctgagctta ttgatcatgc tggtttggat
      961 tcagttgttt atctccggat ttactggctg gggcttaaga tctttactcc aatagcagtg
     1021 cttgcttggg cagttcttgt gccagtcaac tggactaata acacattgga gatggctaag
     1081 cagttaagga atgtaacttc gagcgacatt gacaaactct ctgtttcaaa tattccagag
     1141 tattcaatga ggttttggac tcatatagta atggcttatg cctttaccat ctggacttgt
     1201 tatgtgctga tgaaagaata tgagacaatt gctaacatga ggctccagtt tgttgcatca
     1261 gaagctcgtc gacctgacca gttcactgtc cttgttagga atgtacctcc ggacgcagat
     1321 gaatctgtaa gtgaactggt agagcatttt ttcctggtca atcatcctga tcactaccta
     1381 acacatcagg ttgtatgcaa tgcaaacaag ctggccgatt tggtgaaaaa gaagaaaaag
     1441 ctgcagaatt ggcttgatta ctaccagctc aaatacgcta ggaataactc tcagagaatt
     1501 atggtgaagc ttggctttct tgggctatgg ggacaaaaag tcgatgccat tgaacattac
     1561 attgctgaaa ttgacaaaat atcaaaagag atcagtaaag aaagagagga agtggtgaat
     1621 gatcctaagg ccattatgcc agcagcgttt gtctccttta aaacacgatg ggctgctgcg
     1681 gtttgtgctc agactcaaca gacccgaaac ccaacccaat ggctgaccga atgggctcca
     1741 gaaccgcgtg atgtgttttg gtcaaatctt gctattccat atgtttctct gacagtaagg
     1801 aggttgatca tgcatgttgc cttcttcttc ctaaccttct tcttcattgt ccccatcgcg
     1861 tttgttcaat ctcttgctac cattgaaggg attgtgaaag ctgctccgtt cttgaagttt
     1921 attgtagatg ataaattcat gaaatcggtg atacaaggtt tccttccggg tattgcactg
     1981 ctgcttttca aggcctttct gccatccatt ttgatgatca tgtccaaatt tgaaggcttc
     2041 acatcgatct catctttaga gagacgagca gcgtttcgat attacatctt caacttagtg
     2101 aacgtctttc ttgctagcgt tatcgctgga gctgcgtttg aacagcttaa ctctttcctc
     2161 aatcaatccg caaaccaaat ccccaaaacc attggtgtgg cgataccgat gaaagcaact
     2221 ttcttcatca cgtatataat ggttgatggt tgggcaggag ttgcaggaga gattctaatg
     2281 ctgaaaccat tgattatgtt ccatctcaaa aacgccttct tggttaagac tgataaagac
     2341 agagaggaag caatggaccc gggaagcatt ggttttaaca cgggtgagcc tcggatacag
     2401 ctctactttc ttctcggtct tgtctacgct cctgtgacac cgatgcttct tccttttatc
     2461 ttggtcttct tcgcccttgc ttacattgta tatcgccacc aaatcattaa cgtatacaat
     2521 caagaatacg agagtgctgc agcgttttgg ccagacgttc atggacgagt catagcagca
     2581 ttggtaatat cacagttgct tctgatgggt ctattgggaa caaagcacgc tgcattagct
     2641 gcaccgtttc tcattgcttt gcctgtgctt accattggtt tccaccactt ctgcaaaggt
     2701 cgttatgagc cagctttcat cagataccct ttacaggaag ctatgatgaa agatacatta
     2761 gaaaccgcaa gagaaccaaa cctgaacctt aaaggctact tgcaaaatgc ttacgttcat
     2821 ccggttttca aaggcgatga agacgattat gacattgatg acaagctcgg gaagtttgag
     2881 gatgaagcca ttattgttcc caccaaacgt cagtcgagga gaaatactcc ggctcctagc
     2941 ataatcagtg gggacgattc gccgtctttg ccctttagtg gtaaactagt catggtgagc
     3001 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3061 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3121 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3181 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     3241 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3301 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3361 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3421 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3481 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3541 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3601 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3661 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc
     3721 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     3781 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     3841 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3901 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     3961 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     4021 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     4081 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4141 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4201 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4261 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4321 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4381 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     4441 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4501 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     4561 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     4621 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     4681 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4741 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4801 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4861 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     4921 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4981 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5041 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5101 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     5161 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5221 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5281 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5341 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5401 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5461 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5521 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5581 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5641 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5701 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5761 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5821 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5881 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     5941 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     6001 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     6061 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     6121 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     6181 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6241 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6301 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6361 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6421 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6481 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6541 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6601 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6661 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6721 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6781 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6841 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6901 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6961 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7021 tggataaccg tattaccgcc atgcat
//
