LOCUS       sequence-454654-        7454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse TMEM16F-I521A-eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7454)
  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 7454)
  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: TMEM16F-I521A-peGFP-N1
FEATURES             Location/Qualifiers
     source          1..7454
                     /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"
     regulatory      3394..3403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3400..3402,3403..3405,3406..4119)
                     /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(3400..3402,3403..3405,3406..4116)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3445..3466)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3706..3725)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4053..4074
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4242..4363
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4279..4298)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4333..4352
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4370..4825)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4507..4528)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4719..4738
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4852..4956
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4958..5315
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4958..5149
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4981..5001)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5166..5301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5350..6144
                     /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             5350..6144
                     /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(5404..5423)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6014..6033
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6332..6351)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6376..6423
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6752..7340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7241..7260
                     /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 gcgggccacc
      661 atgactagga aggtcctgct gaacatggag ctggaggagg acgacgatga ggatggagac
      721 attgtgctgg aaaactttga ccagacaatt gtctgcccca cctttggatc actggagaat
      781 cagcaggact tcaggactcc agagtttgaa gaatttaacg ggaagcccga ctccctcttt
      841 ttcaccgatg gccagaggcg aatcgacttc atcctcgtgt atgaagatga gagcaaaaag
      901 gagaacaata agaaagggac aaatgagaaa cagaagagga aaagacaagc atacgaatct
      961 aaccttatct gccatgggct gcagctggaa gcaacaagat ctgtttctga tgacaagctt
     1021 gtgttcgtaa aagtgcacgc gccctgggaa gtgctgtgca cctatgctga gatcatgcac
     1081 atcaaactcc cgctaaagcc aaacgacctg aaaacgcgct cgccctttgg caacctcaac
     1141 tggttcacca aggtcctccg ggtgaacgag agtgtcatca agccagagca ggagttcttc
     1201 actgcccctt ttgagaagag ccggatgaat gatttctaca tcctcgatag agattccttc
     1261 ttcaaccctg ccaccagaag ccgcattgtt tatttcatcc tctctcgggt caaataccaa
     1321 gtgatgaaca acgttaacaa atttgggatt aatagactgg tcagctctgg aatctacaaa
     1381 gcagcgtttc ctctgcacga ctgcagattc aactatgagt cggaggacat cagttgtcct
     1441 agcgagcgtt acctcctgta cagagaatgg gctcaccctc ggagtatata caagaagcag
     1501 cccttggatc ttatcaggaa gtattacggc gagaagattg gaatctactt tgcttggctg
     1561 ggctattaca cgcagatgct ccttctcgca gctgtggtgg gcgtggcctg cttcctctat
     1621 ggatatcttg atcaggataa ctgcacttgg agcaaagagg tctgtgatcc tgacatcggt
     1681 ggccagatcc tgatgtgccc ccagtgtgac aggctgtgtc cgttctggag gctgaatatc
     1741 acctgtgaat cttctaagaa attgtgcatc tttgacagtt ttggaaccct gatcttcgct
     1801 gtatttatgg gagtgtgggt tacattgttt ttggagtttt ggaagcggcg ccaggcagag
     1861 ctcgaatatg aatgggacac cgttgagcta cagcaagagg aacaggcccg gccagagtat
     1921 gaggcccagt gcaatcacgt ggtgataaac gagatcactc aggaggagga gcgcatccca
     1981 tttaccacct gtgggaagtg catccgggtc acgctgtgtg cgagcgccgt cttcttctgg
     2041 atcctgctca tcatcgcctc tgtgatcggg atcattgtct acaggctgtc cgtgttcatc
     2101 gtattttcca caacgctccc caagaacccc aacgggacag acccgatcca gaagtacctg
     2161 accccacaga tggccacatc catcacagcc tccatcatca gcttcatcat cgccatgatc
     2221 ctcaacacga tctacgagaa ggtggccatc atgatcacca acttcgagct cccaaggacc
     2281 cagacggatt atgagaacag cctgaccatg aagatgttct tgttccagtt tgtcaactat
     2341 tactcctcat gcttctacat cgcattcttc aagggcaaat tcgtgggcta tcccggggac
     2401 ccagtgtact tgctgggcaa atacagaagc gaagagtgtg acccgggggg ctgcctcctt
     2461 gaactgacca cacagctgac gatcatcatg gggggaaagg caatctggaa caacatacaa
     2521 gaagtcttgc tcccatgggt tatgaatcta attggacgat ataaaagagt ctcgggatca
     2581 gaaaagataa ccccacgatg ggaacaggat taccatctgc agcccatggg caagctggga
     2641 ttgttctatg agtatcttga aatgattatt cagtttgggt tcgtcacttt atttgtggcc
     2701 tctttcccac tggctcccct gctggctctg gtgaacaata ttttggaaat aagagtggac
     2761 gcgtggaagc tcacaaccca gtttaggcgc atggtgcccg aaaaagccca ggatatcggc
     2821 gcgtggcagc ccatcatgca aggaatagcc atcctggccg tggtgaccaa cgccatgatc
     2881 attgctttca catcagacat gatcccccgc ctggtgtatt attggtcttt ctctatccct
     2941 ccctacgggg accacactta ttacaccatg gacggctaca tcaacaacac tctctctgtc
     3001 ttcaacatca cggacttcaa gaacacagac aaagaaaacc catacattgg gcttggtaac
     3061 tataccttgt gcaggtaccg tgacttccga aacccacctg ggcacccaca agagtataaa
     3121 cacaacatct actattggca cgtgattgcc gccaagctgg ccttcatcat tgtcatggag
     3181 cacatcatct actctgtgaa gttcttcatt tcctacgcaa ttccagatgt gtccaaaatc
     3241 acgaaaagca agatcaagag ggagaaatac ctcacgcaga agttgcttca cgagagtcac
     3301 ctcaaagacc tgaccaaaaa catggggatc atcgccgaga ggataggagg aacggttgac
     3361 aacagcgtgc ggccaaaact cgaagcaccg gtcgccacca tggtgagcaa gggcgaggag
     3421 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3481 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     3541 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     3601 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3661 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     3721 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3781 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3841 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     3901 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3961 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4021 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4081 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca
     4141 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     4201 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     4261 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4321 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     4381 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     4441 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     4501 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     4561 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4621 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4681 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4741 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4801 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4861 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4921 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     4981 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5041 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     5101 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     5161 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5221 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     5281 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     5341 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     5401 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     5461 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     5521 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     5581 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     5641 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     5701 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5761 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5821 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     5881 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5941 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     6001 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     6061 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     6121 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     6181 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     6241 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     6301 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     6361 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     6421 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     6481 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     6541 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     6601 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6661 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6721 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6781 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6841 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6901 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6961 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7021 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7081 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7141 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7201 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7261 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7321 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7381 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7441 ttaccgccat gcat
//
