LOCUS       sequence-454523-        7744 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses mouse TMEM16A-I547K-eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7744)
  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 7744)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: TMEM16A-I547K-peGFP-N1
FEATURES             Location/Qualifiers
     source          1..7744
                     /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"
     CDS             complement(3216..3227)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     regulatory      3684..3693
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3690..3692,3693..3695,3696..4409)
                     /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(3690..3692,3693..3695,3696..4406)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3735..3756)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3996..4015)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4343..4364
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4532..4653
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4569..4588)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4623..4642
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4660..5115)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4797..4818)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5009..5028
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5142..5246
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5248..5605
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5248..5439
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5271..5291)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5456..5591
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5640..6434
                     /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             5640..6434
                     /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(5694..5713)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6304..6323
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6622..6641)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6666..6713
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7042..7630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7531..7550
                     /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 attcggcacg aggcagcgac atcggcctgg
      661 agggcttgac cccagaggga acctctgcgg accgagagtg ccagcgggga ccggaaacaa
      721 tcgcgcacga ggcacaggac gcaggaaccc ccaactcagg tgacgccacc ggggttgtgg
      781 atggggagcg cgaggccacc atgagggtcc ccgagaagta ctcgacgctc ccggcggagg
      841 accgcagcgt ccacatcgtg aacatctgcg ccatcgagga cctgggctac ctgccgtccg
      901 agggcacgtt gctgaactct ctgtccgtgg accccgacgc cgaatgcaag tatggactat
      961 acttcaggga tggcaaacgg aaggtggact acatcttggt ataccatcac aagagagcct
     1021 cagggagcag gactctggcc aggaggggac tacaaaatga catggtcctg gggacccgca
     1081 gcgtcaggca ggaccagccc cttcccggga aggggagccc tgtggatgca ggctcaccgg
     1141 aagtccccat ggattaccat gaagatgaca aacgcttcag acgggaggaa tatgagggca
     1201 acctgttgga ggcaggcctg gagttggaga atgacgagga taccaaaatc catggtgtcg
     1261 ggtttgtgaa gatccatgcg ccctggcatg tgctctgtag ggaagctgag tttttgaaac
     1321 taaagatgcc cacaaagaag gtgtaccaca tcagtgagac gcgaggcctc ctgaaaacca
     1381 tcaactcggt tctgcagaag atcacagacc ccatccagcc caaggtggct gagcacaggc
     1441 cacagaccac aaagaggctc tcctatccct tctcccggga gaagcaacac ctattcgacc
     1501 tgactgacag ggactctttt ttcgacagca aaacccggag cacaatagtc tatgaaatcc
     1561 tgaagagaac aacgtgcacc aaggccaagt acagcatggg tatcaccagc ctcctggcca
     1621 atggcgtata ctcagctgca taccctctgc acgatgggga ctatgagggt gacaacgttg
     1681 agttcaacga caggaaactc ctgtatgagg aatgggcaag ttacggagtc ttctacaaat
     1741 accagcccat tgacctggtc aggaaatact ttggtgagaa ggttggcctg tactttgcct
     1801 ggcttggagc ctacacccag atgctcatcc ctgcctcgat cgtgggtgtc attgtctttc
     1861 tctatggatg tgccactgtg gacgaaaaca tccccagtat ggagatgtgt gaccagagat
     1921 acaacatcac catgtgtcct ctgtgtgaca agacctgcag ctactggaag atgagctcag
     1981 cctgtgccac agcccgtgcc agtcaccttt ttgataaccc tgccaccgtc ttcttctctg
     2041 tgtttatggc cctctgggct gccactttca tggagcactg gaaacggaag cagatgaggc
     2101 tcaactaccg atgggacctc acaggcttcg aggaggagga ggatcatccc agagcagagt
     2161 atgaagccag agtcttagag aagtcactga gaaaagaatc cagaaacaaa gagaccgaca
     2221 aggtgaagct gacctggagg gaccgattcc cagcctattt caccaatctt gtctccatca
     2281 tcttcatgat cgcagtgaca tttgcaatcg tcctcggagt tatcatctat agaatctcca
     2341 cagctgcagc cttggccatg aactcctccc cgtctgtgcg gtccaacatc cgggttacag
     2401 tcacggccac cgctgttatc atcaacctcg tggtcatcaa gctgctggat gaagtttacg
     2461 gctgcattgc caggtggctc accaagattg aggtcccaaa gacagagaag agctttgagg
     2521 agaggctaac cttcaaggcc ttcctgctca agtttgtgaa ctcttacact cccatcttct
     2581 atgtcgcctt cttcaaaggc cggtttgttg gtcggcccgg tgactacgtg tacatcttcc
     2641 gctctttccg gatggaggag tgtgccccgg gcggctgcct catggagctc tgtatccagc
     2701 tgagcatcat tatgctgggc aagcagctaa tccagaacaa tctcttcgag attggcatcc
     2761 cgaagatgaa aaagttcatc cgctacctga agctgcgcag acagagcccc tcagaccgtg
     2821 aagagtacgt gaagcggaag cagcgctatg aggtggactt caacctcgaa cctttcgccg
     2881 gcctcacgcc cgagtacatg gaaatgatca ttcagttcgg ctttgtcacc ctgtttgttg
     2941 cgtccttccc tctggctcca ctcttcgccc tgctaaacaa catcattgag atccgcctgg
     3001 atgccaaaaa gtttgtcacc gagctacgga ggccagtagc catcagagcc aaagacatcg
     3061 gcatctggta taacatcctc agaggtgttg ggaagctggc tgtcatcatt aatgcctttg
     3121 tgatctcctt cacgtctgac ttcatccctc gcctggtgta cctctacatg tacagtcaga
     3181 atgggaccat gcacggcttc gtcaatcaca cgctctcttc cttcaatgtc agcgacttcc
     3241 agaatggcac agcacccaat gacccactgg acctgggcta tgaggttcag atctgcaggt
     3301 ataaagatta ccgggaaccc ccatggtcag aacacaagta tgacatctcc aaagacttct
     3361 gggctgtcct ggccgcccga ctggcatttg tcattgtctt ccagaacctg gtgatgttca
     3421 tgagtgactt tgtggactgg gtgatccctg atatccccaa agacatcagc cagcagatcc
     3481 acaaagagaa ggttctcatg gtggagctgt ttatgcgtga ggagcagggc aagcagcagc
     3541 tactggacac atggatggag aaggagaagc caagggatgt gccttgtaac aaccacagcc
     3601 ccacaaccca cccagaggca ggcgacggca gcccagtccc cagctacgag taccatgggg
     3661 acgcgctgcg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg
     3721 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3781 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3841 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3901 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3961 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     4021 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4081 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4141 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4201 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4261 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4321 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4381 ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca
     4441 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     4501 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     4561 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4621 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     4681 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     4741 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4801 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4861 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4921 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4981 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5041 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5101 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5161 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5221 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     5281 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5341 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     5401 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     5461 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     5521 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     5581 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     5641 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     5701 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     5761 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5821 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     5881 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     5941 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     6001 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     6061 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     6121 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     6181 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     6241 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     6301 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     6361 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     6421 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     6481 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     6541 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     6601 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     6661 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     6721 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     6781 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     6841 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     6901 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6961 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7021 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7081 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7141 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7201 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7261 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7321 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7381 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7441 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7501 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7561 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7621 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7681 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     7741 gcat
//
