LOCUS       sequence-358930-        6697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the FRET membrane voltage sensor Mermaid in vertebrate 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  TITLE     CAG-Mermaid in Tol2
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6697)
  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: 
            "Unpublished"
COMMENT     Sequence Label: CAG-Mermaid in Tol2
FEATURES             Location/Qualifiers
     source          1..6697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..72
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     enhancer        79..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     537..557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     gap             563..585
                     /estimated_length=23
     primer_bind     943..962
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1018..1027
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1780..2457
                     /codon_start=1
                     /product="monomeric variant of Umikinoko-Green fluorescent 
                     protein"
                     /label=mUkG1
                     /translation="MSVIKEEMKIKLHMEGNVNGHAFVIEGDGKGKPYDGTQTLNLTVK
                     EGAPLPFSYDILTNAFQYGNRAFTKYPADIPDYFKQTFPEGYSWERTMSYEDNAICNVR
                     SEISMEGDCFIYKIRFDGKNFPPNGPVMQKKTLKWEPSTEMMYVRDGFLMGDVNMALLL
                     EGGGHHRCDFKTSYKAKKVVQLPDAHKIDHRIEILSHDRDYSKVKLYENAVARNSLLPS
                     QASK"
     CDS             2467..3123
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    3137..3271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3174..3193)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3228..3247
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3279..3334
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     misc_feature    3344..3415
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     protein_bind    complement(3416..3449)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(3507..3694,3695..3706)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3787..3803)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3787..3803)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3800..3822)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3811..3827
                     /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(3835..3841,3842..3859,3860..3865))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3880..3901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4018..4035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4189..4777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4269..4288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4948..5739,5740..5808))
                     /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     5571..5590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5809..5913)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5981..5999
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6037..6059)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6159..6178
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6372..6394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6386..6403
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6387..6403
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(6480..6491,6492..6654)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 catcttggac cattagctcc acaggtatct tcttccctct agtggtcata acagcagctt
       61 cagctacctc tccctaggcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      421 ctcccccccc tccccacccc ywmywwkkyw yywmyyyatt kwykwmwwwk kkwgtkyrky
      481 kwkgsrssmr rrksrrnggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      541 aatgggcggt aggcgtgtac ggnnnnnnnn nnnnnnnnnn nnnnnggtgc ggcggcagcc
      601 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc
      661 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga cgctgccttc gccccgtgcc
      721 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      781 ggtgagcggg cgggacggcc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg
      841 cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggagct agagcctctg
      901 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
      961 tgctgtctca tcattttggc aaagaatttt ggaactctaa ttctccaaat tcaagcagcc
     1021 accatggagg gattcgacgg ttcagatttt agtcctccag ctgatttagt tggcgttgac
     1081 ggtgcagtca tgcggaacgt cgttgacgtc acgataaatg gtgacgtcac tgctccgccg
     1141 aaagctgcgc caagaaaaag tgaatcggta aagaaagttc attggaatga tgtagaccaa
     1201 ggaccgagtg aaaaaccaga gacaagacag gaggaacgaa tagatatacc cgagatttca
     1261 ggtctatggt ggggcgagaa tgaacatgga gtggacgatg ggagaatgga gatacctact
     1321 actggtgtag gtcgcgtcca gtttcgtgtc cgagcagtga ttgatcatct agggatgcga
     1381 gtctttggag tcttcctaat tttcttggac atcatcctca tgatcattga tctcagtctt
     1441 ccaggaaaaa gtgaatcttc ccaatccttt tatgacggga tggctttggc tctttcttgt
     1501 tatttcatgc tggatttagg attaaggata tttgcatacg ggcccaagaa tttcttcacc
     1561 aacccctggg aggttgctga tggtttgatt atcgttgtca cattcgtcgt cacgatattt
     1621 tacactgtgt tagatgaata cgtgcaagaa acaggagccg atggtttggg gcagttggtt
     1681 gtgttggccc gtttgctgcg tgtggttcga ttagcaagaa tattttattc ccatcaacaa
     1741 atgaaggctt caagcagaag aacaatatca ggtgatccta tgagtgtgat taaagaagaa
     1801 atgaagatca agctgcacat ggaaggaaat gtaaacggtc atgcatttgt gattgaagga
     1861 gatggaaaag gaaagcctta cgatgggaca cagactttaa acctgacagt gaaagaaggc
     1921 gcacctctcc ctttttctta cgacatcttg acaaatgcgt tccagtacgg aaatagagca
     1981 ttcactaaat atccagccga tataccagac tatttcaagc agacgtttcc cgaggggtat
     2041 tcatgggaaa gaaccatgag ttatgaagac aacgccattt gcaacgtgag aagcgagatc
     2101 agcatggaag gcgactgctt tatctataaa attcggtttg atggcaagaa ctttcccccc
     2161 aatggtccag ttatgcagaa gaaaactttg aagtgggaac catccactga gatgatgtac
     2221 gtgcgtgatg ggtttctgat gggtgatgtg aacatggctc tgttgcttga aggaggtggc
     2281 catcaccgat gtgacttcaa aacttcctac aaagcgaaaa aggttgtgca gttgccagat
     2341 gctcacaaga ttgaccatcg tattgaaatc ttgagccatg acagggatta cagcaaagtc
     2401 aagctgtatg agaatgcggt tgctcgcaat tctttgctgc caagtcaggc ctctaagctt
     2461 gagactatgg tgagcgtgat caagcccgag atgaagatga ggtactacat ggacggctcc
     2521 gtcaatgggc atgagttcac aatcgagggt gagggcacag gcagacctta cgagggacat
     2581 caggagatga cactgcgcgt cacaatggcc gagggcgggc caatgccttt cgccttcgac
     2641 ctggtgtccc acgtgttctg ttacggccac agagttttta ctaaatatcc agaagagatc
     2701 ccagactatt tcaagcaggc ctttcctgag ggcctgtcct gggagaggtc cctggagttc
     2761 gaggacggcg gctccgcctc cgtgagcgcc cacatcagcc tgaggggcaa caccttctac
     2821 cacaagtcca agttcaccgg cgtgaacttc cccgccgacg gccccatcat gcagaaccag
     2881 agcgtggact gggagccctc caccgagaag atcaccgcca gcgacggcgt gctgaagggc
     2941 gacgtgacca tgtacctgaa gctggagggc ggcggcaacc acaagtgcca gttcaagacc
     3001 acctacaagg ccgccaagga gatcctggag atgcccggcg atcactacat cggccacagg
     3061 ctggtgagga agaccgaggg caacatcacc gagcaggtgg aggacgccgt ggcccactcc
     3121 taagaacaac tagtaaaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3181 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3241 actcatcaat gtatcttatc atgtctggat caggtaccaa taaaggaaat ttattttcat
     3301 tgcaatagtg tgttggaatt ttttgtgtct ctcactgcag taccattcct ttaatggtct
     3361 tttctggaca ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaataac
     3421 ttcgtatagc atacattata cgaagttatg attccttgag caagcgcaat tcaattggtt
     3481 tggtaatagc aagggaaaat agaatgaagt gatctccaaa aaataagtac tttttgactg
     3541 taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta attaagtaaa
     3601 agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa aaataatact
     3661 taagtacagt aatcaagtaa aattactcaa gtactttaca cctctggttc ttgaccccct
     3721 accttcagca agcccagcat ctagagtcga ctgcagaggc ctgcatgcaa gcttggcgta
     3781 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3841 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3901 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3961 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4021 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4081 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4141 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4201 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4261 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4321 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4381 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4441 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4501 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4561 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4621 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4681 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4741 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4801 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4861 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4921 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4981 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5041 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag atccacgctc
     5101 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5161 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5221 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5281 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5341 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5401 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5461 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5521 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5581 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5641 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5701 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5761 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5821 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5881 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5941 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6001 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     6061 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     6121 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     6181 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     6241 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     6301 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     6361 gccagggttt acccagtcac gacgttgtaa aacgacggcc agtgaattct gaatatctgt
     6421 tcagacaccc atatccactc tgttccacac aggtcagagg tttgtccagg agttcttgac
     6481 agaggtgtaa aaagtactca aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa
     6541 ttttactcaa ttaaaagtaa aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa
     6601 gtactccatt aaaattgtac ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa
     6661 ctagagattc ttgtttaagc ttgctagcag atatcat
//
