LOCUS Exported 6697 bp ds-DNA circular SYN 11-APR-2022 DEFINITION Expresses the FRET membrane voltage sensor Mermaid in vertebrate cells. ACCESSION . VERSION . KEYWORDS CAG-Mermaid in Tol2 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DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6697) TITLE CAG-Mermaid in Tol2 JOURNAL Unpublished REFERENCE 2 (bases 1 to 6697) AUTHORS .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 Apr 11, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6697 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 3416..3449 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." 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(3835..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(4948..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" 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 //