LOCUS Exported 8039 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses a tandem GFP-APEX2 fusion to the C-terminus of connexin43 in mammalian cells. ACCESSION . VERSION . KEYWORDS pcDNA3 Connexin43-GFP-APEX2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8039) AUTHORS Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY TITLE Directed evolution of APEX2 for electron microscopy and proximity labeling. JOURNAL Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179. PUBMED 25419960 REFERENCE 2 (bases 1 to 8039) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8039 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 917..926 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2090..2806 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2135..2156) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2396..2415) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2743..2764 /label=EGFP-C /note="EGFP, forward primer" CDS 2846..3592 /codon_start=1 /product="soybean ascorbate peroxidase, engineered to serve as a monomeric tag for electron microscopy and live-cell proteomics (Lam et al., 2015)" /label=APEX2 /note="more active than the original APEX due to the A134P mutation" /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK YAADEDAFFADYAEAHQKLSELGFADA" primer_bind complement(3633..3650) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3639..3863 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3909..4337 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3996..4015) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4206..4227 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4346..4366) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4351..4680 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4531..4666 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4593..4612 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4747..5541 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4801..4820) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5411..5430 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5715..5836 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5752..5771) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5806..5825 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5885..5901) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5885..5901) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5898..5920) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5909..5925 /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(5933..5963) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5978..5999 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6116..6133) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6287..6872) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6367..6386) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7043..7903) /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 7666..7685 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7904..8008) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 gtttaaactt aagcttgcca ccatgggtga ctggagtgcc ttggggaaat tactggacaa 961 ggtccaagcc tactccaccg ctggagggaa ggtgtggctg tcagtgctct tcatattcag 1021 aatcctgctc ctggggacag ctgttgagtc agcttggggt gatgaacagt ctgcctttcg 1081 ctgtaacact caacaacctg gctgcgaaaa cgtctgctat gacaagtcct tccccatctc 1141 tcacgtgcgc ttctgggtcc ttcagatcat attcgtgtct gtgcccacac tcctgtactt 1201 ggcccatgtg ttctatgtga tgaggaagga agagaagcta aacaagaaag aagaggagct 1261 caaagtggcc cagactgacg gggtcaacgt ggagatgcac ctgaagcaga ttgaaatcaa 1321 gaagttcaag tacgggattg aagagcacgg caaggtgaaa atgaggggcg gcttgctgag 1381 aacctacatc atcagcatcc tcttcaagtc tgtcttcgag gtggccttcc tgctcatcca 1441 gtggtacatc tatgggttca gcttgagcgc ggtctacacc tgcaagagag atccctgccc 1501 ccaccaggta gactgcttcc tctcacgtcc cacggagaaa accatcttca tcatcttcat 1561 gctggtggtg tccttggtgt ctctcgcttt gaacatcatt gagctcttct acgtcttctt 1621 caagggcgtt aaggatcgcg tgaagggaag aagcgatcct taccacgcca ccactggccc 1681 actgagccca tcaaaagact gcggatctcc aaaatacgcc tacttcaatg gctgctcctc 1741 accaacggct ccactctcgc ctatgtctcc tcctgggtac aagctggtta ctggtgacag 1801 aaacaattcc tcgtgccgca attacaacaa gcaagctagc gagcaaaact gggcgaacta 1861 cagcgcagag caaaatcgca tggggcaggc cggaagcacc atctccaact cgcacgccca 1921 gccgttcgat ttccccgacg acaaccagaa tgccaaaaaa gttgctgctg gacatgaact 1981 ccagccatta gccatcgtgg accaacgacc ttccagcaga gccagcagcc gcgccagcag 2041 caggcctcgg cctgatgacc tggagattgg atccgggggt ggtgggtcta tggtgagcaa 2101 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 2161 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 2221 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 2281 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 2341 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 2401 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 2461 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 2521 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc 2581 caacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 2641 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 2701 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 2761 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggggc gcgccaaggg 2821 ctcgggctcg acctcgggct cgggcggaaa gtcttaccca actgtgagtg ctgattacca 2881 ggacgccgtt gagaaggcga agaagaagct cagaggcttc atcgctgaga agagatgcgc 2941 tcctctaatg ctccgtttgg cattccactc tgctggaacc tttgacaagg gcacgaagac 3001 cggtggaccc ttcggaacca tcaagcaccc tgccgaactg gctcacagcg ctaacaacgg 3061 tcttgacatc gctgttaggc ttttggagcc actcaaggcg gagttcccta ttttgagcta 3121 cgccgatttc taccagttgg ctggcgttgt tgccgttgag gtcacgggtg gacctaaggt 3181 tccattccac cctggaagag aggacaagcc tgagccacca ccagagggtc gcttgcccga 3241 tcccactaag ggttctgacc atttgagaga tgtgtttggc aaagctatgg ggcttactga 3301 ccaagatatc gttgctctat ctgggggtca cactattgga gctgcacaca aggagcgttc 3361 tggatttgag ggtccctgga cctctaatcc tcttattttc gacaactcat acttcacgga 3421 gttgttgagt ggtgagaagg aaggtctcct tcagctacct tctgacaagg ctcttttgtc 3481 tgaccctgta ttccgccctc tcgttgacaa atatgcagcg gacgaagatg ccttctttgc 3541 tgattacgct gaggctcacc aaaagctttc cgagcttggg tttgctgatg cctaactcga 3601 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc 3661 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3721 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3781 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3841 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 3901 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3961 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 4021 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 4081 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 4141 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4201 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 4261 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 4321 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 4381 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 4441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 4501 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 4561 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct 4621 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 4681 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg 4741 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 4801 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 4861 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 4921 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 4981 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 5041 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 5101 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 5161 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 5221 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 5281 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 5341 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 5401 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 5461 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 5521 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 5581 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 5641 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 5701 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 5761 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 5821 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 5881 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 5941 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 6001 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 6061 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 6121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 6181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 6241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 6301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 6421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 6481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6721 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt gtttgcaagc 6841 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6901 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 6961 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 7021 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 7081 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 7141 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 7201 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 7261 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 7321 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 7381 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 7441 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 7501 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 7561 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 7621 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 7681 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 7741 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 7801 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 7861 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 7921 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 7981 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc //