LOCUS       sequence-218925-        5451 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 5451)
  TITLE     BK channel plasmids
REFERENCE   2  (bases 1 to 5451)
  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..5451
                     /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      1391..1400
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1397..1399,1400..1402,1403..2116)
                     /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(1397..1399,1400..1402,1403..2113)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1442..1463)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1703..1722)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2050..2071
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2239..2360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2276..2295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2330..2349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2367..2822)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2504..2525)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2716..2735
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2849..2953
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2955..3312
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2955..3146
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2978..2998)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3163..3298
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3347..4141
                     /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             3347..4141
                     /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(3401..3420)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4011..4030
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4329..4348)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4373..4420
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4749..5337
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5238..5257
                     /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 ttgatatcga attccaccat ggcgaagctc cgggtggctt acgagtacac
      781 ggaagccgag gacaagagca tccggctcgg cttgtttctc atcatctccg gcgtcgtgtc
      841 gctcttcatc ttcggcttct gctggctgag tcccgcgctg caggatctgc aagccacgga
      901 ggccaattgc acggtgctgt cggtgcagca gatcggcgag gtgttcgagt gcaccttcac
      961 ctgtggcgcc gactgcaggg gcacctcgca gtacccctgc gtccaggtct acgtgaacaa
     1021 ctctgagtcc aactctaggg cgctgctgca cagcgacgag caccagctcc tgaccaaccc
     1081 caagtgctcc tatatccctc cctgtaagag agaaaatcag aagaatttgg aaagtgtcat
     1141 gaattggcaa cagtactgga aagatgagat tggttcccag ccatttactt gctattttaa
     1201 tcaacatcaa agaccagatg atgtgcttct gcatcgcact catgatgaga ttgtcctcct
     1261 gcattgcttc ctctggcccc tggtgacatt tgtggtgggc gttctcattg tggtcctgac
     1321 catctgtgcc aagagcttgg cggtcaaggc ggaagccatg aagaagcgca agttctcgga
     1381 tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1441 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1501 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1561 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1621 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1681 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1741 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1801 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1861 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1921 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1981 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2041 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2101 cgagctgtac aagtaaagcg gccgcgactc tagatcataa tcagccatac cacatttgta
     2161 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     2221 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2281 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     2341 aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     2401 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2461 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2521 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2581 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2641 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2701 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2761 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2821 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2881 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2941 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     3001 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3061 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3181 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3241 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3301 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3361 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3421 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3481 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     3541 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3601 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3661 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3721 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3781 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3841 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     3901 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3961 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4021 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4081 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4141 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4201 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4261 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     4321 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     4381 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     4441 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     4501 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     4561 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     4621 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4681 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4741 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4801 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4861 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4921 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4981 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5041 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5101 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5161 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5221 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5281 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5341 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5401 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
