LOCUS       sequence-257444-        6346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6346)
  AUTHORS   Green MF, Scott JW, Steel R, Oakhill JS, Kemp BE, Means AR
  TITLE     Ca2+/Calmodulin-dependent Protein Kinase Kinase {beta} Is Regulated 
            by Multisite Phosphorylation.
  JOURNAL   J Biol Chem. 2011 Aug 12;286(32):28066-79. Epub 2011 Jun 13.
  PUBMED    21669867
REFERENCE   2  (bases 1 to 6346)
  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: "J Biol 
            Chem."; date: "2011-08-12"; volume: "286(32)"; pages: "28066-79. 
            Epub 2011 Jun 13"
COMMENT     Sequence Label: pEGFP-N1-CaMKKbeta
FEATURES             Location/Qualifiers
     source          1..6346
                     /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"
     CDS             600..623
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2292..2294,2295..2297,2298..3011)
                     /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(2292..2294,2295..2297,2298..3008)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2598..2617)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2945..2966
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3134..3255
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3171..3190)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3225..3244
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3262..3717)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3399..3420)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3611..3630
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3744..3848
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3850..4207
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3850..4041
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3873..3893)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4058..4193
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4242..5036
                     /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             4242..5036
                     /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(4296..4315)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4906..4925
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5224..5243)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5268..5315
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5644..6232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6133..6152
                     /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 gctagcatgg
      601 actacaagga cgacgatgac aaagctagct catcatgtgt ctctagccag cccaccagcg
      661 accgggtggc cccccaggat gagctgggaa gtgggggtgg cagccgggaa ggccagaagc
      721 cctgtgaggc actgcgggga ctctcatcct taagtatcca cttgggcatg gaatccttca
      781 tcgtggtcac cgagtgtgag ccaggccggg gtgtggacct caacctggcc agagaccagc
      841 ctccggaggc cgatggccag gaactccccc tcgaagcctc ggaccctgag tcccggtccc
      901 cgctttctgg tcgcaagatg tccctgcagg agccgtccca gggcgggccg gcatccagca
      961 gcaacagcct ggacatgaat ggacgctgca tctgcccatc cctgtcctac tcaccagcca
     1021 gctccccaca gtcctctccc cggatgcccc ggcggcccac agtagagtca caccacgtct
     1081 ccattaccgg tttgcaggac tgcgtgcagc tgaatcagta caccctgaag gatgaaattg
     1141 gaaagggctc ctatggtgtt gtcaagctgg cctacaatga aaatgacaat acttattacg
     1201 caatgaaagt gctgtccaaa aagaagctga tccgacaggc tggcttccca cgtcgccccc
     1261 caccacgagg agctcgccca gccccagggg gctgcatcca gcccaggggc cccatcgagc
     1321 aagtgtacca ggagattgct atcctcaaaa agctggatca tcccaacgtg gtgaagctgg
     1381 tagaggtcct ggatgaccct aatgaggacc atctgtacat ggtgtttgag ttggtgaacc
     1441 aagggcctgt aatggaagtg cccaccctca agccactgtc cgaagaccag gcccgcttct
     1501 acttccagga tctgatcaaa ggcatagagt acttgcatta ccagaagatc atccatcggg
     1561 acatcaaacc ttccaacctc ctggtggggg aagacgggca catcaagatt gccgacttcg
     1621 gtgtaagcaa cgagttcaag ggcagcgacg ccttgctgtc taacaccgtg ggcacacctg
     1681 ccttcatggc acccgagtcg ctctcagaga cccgcaagat cttctccgga aaggcgttgg
     1741 atgtttgtgc catgggtgtg acgctgtact gctttgtctt tggccagtgc cctttcatgg
     1801 atgaacgaat catgtgtttg cacagtaaga tcaagagtca ggccctggag tttcccgacc
     1861 agcccgatat agccgaagac ttgaaagatc tgatcactcg gatgttggac aaaaatccag
     1921 agtccaggat tgtggtgcct gaaatcaagc tgcacccttg ggttacgagg cacggggccg
     1981 agccattgcc gtcggaggac gagaactgca cactggtcga ggtgaccgaa gaggaggtcg
     2041 agaattcagt caaacacatt cccagcctgg caactgtgat cctggtaaag accatgattc
     2101 gaaagcgctc attcgggaac ccgttcgaag gtagccgccg ggaggaacgt tccctgtcag
     2161 cgcctggaaa cctgctcacg aagcaaggca gcgaagacag cccccggggc ccagagccgg
     2221 cccctgtggg ggaggaggaa gtgctcttgg cagtcgacgg taccgcgggc ccgggatcca
     2281 ccggtcgcca catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2341 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     2401 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2461 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2521 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2581 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2641 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2701 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2761 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2821 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2881 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2941 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     3001 tgtacaagta aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt
     3061 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     3121 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3181 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3241 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     3301 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     3361 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     3421 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     3481 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     3541 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     3601 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     3661 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     3721 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3781 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3841 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     3901 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3961 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4021 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4081 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4141 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4201 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4261 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4321 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4381 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     4441 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4501 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4561 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4621 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4681 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4741 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     4801 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4861 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4921 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4981 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     5041 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     5101 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5161 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     5221 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     5281 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     5341 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5401 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     5461 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     5521 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5581 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5761 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6301 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
