LOCUS       sequence-203602-        8042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a tandem GFP-APEX2 fusion to the C-terminus of connexin43 
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8042)
  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 8042)
  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: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT     Sequence Label: pcDNA3 Connexin43-GFP-APEX2
FEATURES             Location/Qualifiers
     source          1..8042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        167..546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        547..750
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     701..721
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     795..814
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        795..813
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      849..858
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2022..2024,2025..2027,2028..2738)
                     /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(2067..2088)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2328..2347)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2675..2696
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2778..3524
                     /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(3565..3582)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3571..3795
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3841..4269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3928..3947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4138..4159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4278..4298)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4283..4612
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4463..4598
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4525..4544
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4679..5473
                     /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(4733..4752)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5343..5362
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5647..5768
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5684..5703)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5738..5757
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5817..5833)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5817..5833)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5830..5852)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5841..5857
                     /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(5865..5871,5872..5889,5890..5895))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5910..5931
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6048..6065)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6219..6807)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6299..6318)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6978..7769,7770..7838))
                     /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     7601..7620
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7839..7943)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8018..8037)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
       61 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
      121 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      181 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      241 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      301 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      361 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      421 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      481 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      541 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      601 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      661 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      721 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
      781 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcgtttaaac
      841 ttaagcttgc caccatgggt gactggagtg ccttggggaa attactggac aaggtccaag
      901 cctactccac cgctggaggg aaggtgtggc tgtcagtgct cttcatattc agaatcctgc
      961 tcctggggac agctgttgag tcagcttggg gtgatgaaca gtctgccttt cgctgtaaca
     1021 ctcaacaacc tggctgcgaa aacgtctgct atgacaagtc cttccccatc tctcacgtgc
     1081 gcttctgggt ccttcagatc atattcgtgt ctgtgcccac actcctgtac ttggcccatg
     1141 tgttctatgt gatgaggaag gaagagaagc taaacaagaa agaagaggag ctcaaagtgg
     1201 cccagactga cggggtcaac gtggagatgc acctgaagca gattgaaatc aagaagttca
     1261 agtacgggat tgaagagcac ggcaaggtga aaatgagggg cggcttgctg agaacctaca
     1321 tcatcagcat cctcttcaag tctgtcttcg aggtggcctt cctgctcatc cagtggtaca
     1381 tctatgggtt cagcttgagc gcggtctaca cctgcaagag agatccctgc ccccaccagg
     1441 tagactgctt cctctcacgt cccacggaga aaaccatctt catcatcttc atgctggtgg
     1501 tgtccttggt gtctctcgct ttgaacatca ttgagctctt ctacgtcttc ttcaagggcg
     1561 ttaaggatcg cgtgaaggga agaagcgatc cttaccacgc caccactggc ccactgagcc
     1621 catcaaaaga ctgcggatct ccaaaatacg cctacttcaa tggctgctcc tcaccaacgg
     1681 ctccactctc gcctatgtct cctcctgggt acaagctggt tactggtgac agaaacaatt
     1741 cctcgtgccg caattacaac aagcaagcta gcgagcaaaa ctgggcgaac tacagcgcag
     1801 agcaaaatcg catggggcag gccggaagca ccatctccaa ctcgcacgcc cagccgttcg
     1861 atttccccga cgacaaccag aatgccaaaa aagttgctgc tggacatgaa ctccagccat
     1921 tagccatcgt ggaccaacga ccttccagca gagccagcag ccgcgccagc agcaggcctc
     1981 ggcctgatga cctggagatt ggatccgggg gtggtgggtc tatggtgagc aagggcgagg
     2041 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2101 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2161 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2221 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2281 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2341 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2401 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2461 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gccaacttca
     2521 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2581 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2641 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2701 ccgccgggat cactctcggc atggacgagc tgtacaaggg gcgcgccaag ggctcgggct
     2761 cgacctcggg ctcgggcgga aagtcttacc caactgtgag tgctgattac caggacgccg
     2821 ttgagaaggc gaagaagaag ctcagaggct tcatcgctga gaagagatgc gctcctctaa
     2881 tgctccgttt ggcattccac tctgctggaa cctttgacaa gggcacgaag accggtggac
     2941 ccttcggaac catcaagcac cctgccgaac tggctcacag cgctaacaac ggtcttgaca
     3001 tcgctgttag gcttttggag ccactcaagg cggagttccc tattttgagc tacgccgatt
     3061 tctaccagtt ggctggcgtt gttgccgttg aggtcacggg tggacctaag gttccattcc
     3121 accctggaag agaggacaag cctgagccac caccagaggg tcgcttgccc gatcccacta
     3181 agggttctga ccatttgaga gatgtgtttg gcaaagctat ggggcttact gaccaagata
     3241 tcgttgctct atctgggggt cacactattg gagctgcaca caaggagcgt tctggatttg
     3301 agggtccctg gacctctaat cctcttattt tcgacaactc atacttcacg gagttgttga
     3361 gtggtgagaa ggaaggtctc cttcagctac cttctgacaa ggctcttttg tctgaccctg
     3421 tattccgccc tctcgttgac aaatatgcag cggacgaaga tgccttcttt gctgattacg
     3481 ctgaggctca ccaaaagctt tccgagcttg ggtttgctga tgcctaactc gagtctagag
     3541 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3601 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3661 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3721 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3781 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3841 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3901 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3961 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4021 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4081 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4141 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4201 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4261 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4321 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4381 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4441 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4501 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     4561 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     4621 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4681 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4741 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4801 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4861 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4921 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4981 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     5041 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     5101 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     5161 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     5221 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     5281 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     5341 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     5401 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5461 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5521 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5581 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5641 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5701 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5761 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5821 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5881 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5941 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6001 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6061 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6121 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6181 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6241 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6301 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6361 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6421 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6481 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6541 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6601 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6661 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6721 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6781 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6841 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6901 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6961 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7021 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7081 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7141 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7201 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7261 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7321 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7381 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7441 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7501 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7561 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7621 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7681 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7741 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7801 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7861 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7921 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     7981 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     8041 ag
//
