LOCUS       sequence-214289-        5440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human BK beta4 subunit in C-term translational frame with EGFP 
            (EGFP-N1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5440)
  TITLE     BK channel plasmids
REFERENCE   2  (bases 1 to 5440)
  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"
COMMENT     Sequence Label: BK beta4 EGFP
FEATURES             Location/Qualifiers
     source          1..5440
                     /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"
     promoter        619..637
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           659..701
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      1380..1389
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1386..1388,1389..1391,1392..2105)
                     /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(1386..1388,1389..1391,1392..2102)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1431..1452)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1692..1711)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2039..2060
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2228..2349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2265..2284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2319..2338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2356..2811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2493..2514)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2705..2724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2838..2942
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2944..3301
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2944..3135
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2967..2987)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3152..3287
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3336..4130
                     /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             3336..4130
                     /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(3390..3409)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4000..4019
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4318..4337)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4362..4409
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4738..5326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5227..5246
                     /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 atctcgagaa ttaaccctca ctaaagggaa caaaagctgg gtacccttct
      661 tgttcttttt gcagaagctc agaataaacg ctcaactttg gcagagctag cgtcgacggt
      721 atcgataagc ttccaccatg gcgaagctcc gggtggctta cgagtacacg gaagccgagg
      781 acaagagcat ccggctcggc ttgtttctca tcatctccgg cgtcgtgtcg ctcttcatct
      841 tcggcttctg ctggctgagt cccgcgctgc aggatctgca agccacggag gccaattgca
      901 cggtgctgtc ggtgcagcag atcggcgagg tgttcgagtg caccttcacc tgtggcgccg
      961 actgcagggg cacctcgcag tacccctgcg tccaggtcta cgtgaacaac tctgagtcca
     1021 actctagggc gctgctacac agcgacgagc accagctcct gaccaacccc aagtgctcct
     1081 atatccctcc ctgtaagaga gaaaatcaga agaatttgga aagtgtcatg aattggcaac
     1141 agtactggaa agatgagatt ggttcccagc catttacttg ctattttaat caacatcaaa
     1201 gaccagatga tgtgcttctg catcgcactc atgatgagat tgtcctcctg cattgcttcc
     1261 tctggcccct ggtgacattt gtggtgggcg ttctcattgt ggtcctgacc atctgtgcca
     1321 agagcttggc ggtcaaggcg gaagccatga agaagcgcaa gttctcggat ccaccggtcg
     1381 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1441 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1501 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1561 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1621 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1681 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1741 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1801 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1861 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1921 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1981 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2041 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2101 agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     2161 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2221 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2281 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2341 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2401 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2461 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2521 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2581 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2641 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2701 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2761 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     2821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2941 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3001 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3061 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3121 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3181 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3241 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3301 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3361 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3421 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3481 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3541 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3601 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3661 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3721 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3781 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3841 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     3901 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3961 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4021 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4081 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4141 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4201 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4261 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4321 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4381 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4441 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4501 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4561 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     4621 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4681 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4741 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4801 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4861 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4921 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4981 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5041 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5101 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5161 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5221 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5281 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5341 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5401 atcccctgat tctgtggata accgtattac cgccatgcat
//
