LOCUS       sequence-490426-        7576 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  Mammalian expression of CDKL5 isoform1 (1-960) with C-terminal GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7576)
  AUTHORS   Otani Y, Goodall E, Srinivasan V, Ball NJ, Barnett SFH, Zech T, 
            Saarikangas J, Goult BT
  TITLE     Cyclin-dependent kinase-like 5 (CDKL5) binds to talin and is 
            anchored at the postsynaptic density via direct interaction with PDZ
            domains
  JOURNAL   bioRxiv 2025.08.27.672337; doi: 
            https://doi.org/10.1101/2025.08.27.672337
REFERENCE   2  (bases 1 to 7576)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.08.27.672337; doi: https://doi.org/10.1101/2025.08.27.672337"
COMMENT     Sequence Label: CDKL5(isoform1)-GFP
FEATURES             Location/Qualifiers
     source          1..7576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4219..4228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4225..4227,4228..4230,4231..4944)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4270..4291)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4531..4550)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4878..4899
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5067..5188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5104..5123)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5158..5177
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5195..5650)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5332..5353)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5544..5563
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5677..5781
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5783..6140
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5783..5974
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5806..5826)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5991..6126
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6175..6969
                     /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             6175..6969
                     /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(6229..6248)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6839..6858
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7157..7176)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7201..7248
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
     1321 gacatgaaga ttcctaacat tggtgatgtg atgaataaat ttgagatcct tggggttgta
     1381 ggtgaaggag cctatggagt tgtacttaaa tgcagacaca aggaaacaca tgaaattgtg
     1441 gcgatcaaga aattcaagga cagtgaagaa aatgaagaag tcaaagaaac gactttacga
     1501 gagcttaaaa tgcttcggac tctcaagcag gaaaacattg tggagttgaa ggaagcattt
     1561 cgtcggaggg gaaagttgta cttggtgttt gagtatgttg aaaaaaatat gctcgaattg
     1621 ctggaagaaa tgccaaatgg agttccacct gagaaagtaa aaagctacat ctatcagcta
     1681 atcaaggcta ttcactggtg ccataagaat gatattgtcc atcgagatat aaaaccagaa
     1741 aatctcttaa tcagccacaa tgatgtccta aaactgtgtg actttggttt tgctcgtaat
     1801 ctgtcagaag gcaataatgc taattacaca gagtacgttg ccaccagatg gtatcggtcc
     1861 ccagaactct tacttggcgc tccctatgga aagtccgtgg acatgtggtc ggtgggctgt
     1921 attcttgggg agcttagcga tggacagcct ttatttcctg gagaaagtga aattgaccaa
     1981 ctttttacta ttcagaaggt gctaggacca cttccatctg agcagatgaa gcttttctac
     2041 agtaatcctc gcttccatgg gctccggttt ccagctgtta accatcctca gtccttggaa
     2101 agaagatacc ttggaatttt gaatagtgtt ctacttgacc taatgaagaa tttactgaag
     2161 ttggacccag ctgacagata cttgacagaa cagtgtttga atcaccctac atttcaaacc
     2221 cagagacttc tggatcgttc tccttcaagg tcagcaaaaa gaaaacctta ccatgtggaa
     2281 agcagcacat tgtctaatag aaaccaagcc ggcaaaagta ctgctttgca gtctcaccac
     2341 agatctaaca gcaaggacat ccagaacctg agtgtaggcc tgccccgggc tgacgaaggt
     2401 ctccctgcca atgaaagctt cctaaatgga aaccttgctg gagctagtct tagtccactg
     2461 cacaccaaaa cctaccaagc aagcagccag cctgggtcta ccagcaaaga tctcaccaac
     2521 aacaacatac cacaccttct tagcccaaaa gaagccaagt caaaaacaga gtttgatttt
     2581 aatattgacc caaagccttc agaaggccca gggacaaagt acctcaagtc aaacagcaga
     2641 tctcagcaga accgccactc attcatggaa agctctcaaa gcaaagctgg gacactgcag
     2701 cccaatgaaa agcagagtcg gcatagctat attgacacaa ttccccagtc ctctaggagt
     2761 ccctcctaca ggaccaaggc caaaagccat ggggcactga gtgactccaa gtctgtgagc
     2821 aacctttctg aagccagggc ccaaattgcg gagcccagta ccagtaggta cttcccatct
     2881 agctgcttag acttgaattc tcccaccagc ccaaccccca ccagacacag tgacacgaga
     2941 actttgctca gcccttctgg aagaaataac cgaaatgagg gaacgctgga ctcacgtcga
     3001 accacaacca gacattctaa gacgatgggg gaattgaagc tgccggagca catggacagt
     3061 agccattccc attcactgtc tgcacctcac gaatcttttt cttatggact gggctacacc
     3121 agcccctttt cttcccagca acgtcctcat aggcattcta tgtatgtgac ccgtgacaaa
     3181 gtgagagcca agggcttgga tggaagcttg agcatagggc aagggatggc agctagagcc
     3241 aacagcctgc aactcttgtc accccagcct ggagaacagc tccctccaga gatgactgtg
     3301 gcaagatctt cggtcaaaga gacctccaga gaaggcacct cttccttcca tacacgccag
     3361 aagtctgagg gtggagtgta tcatgaccca cactctgatg atggcacagc ccccaaagaa
     3421 aatagacacc tatacaatga tcctgtgcca aggagagttg gtagctttta cagagtgcca
     3481 tctccacgtc cagacaattc tttccatgaa aataatgtgt caactagagt ttcttctcta
     3541 ccatcagaga gcagttctgg taccaaccat agcaagcggc agcctgcctt cgacccctgg
     3601 aagtcacctg agaacatttc tcatagcgag cagctgaagg aaaaggaaaa gcagggcttc
     3661 ttcaggagta tgaaaaagaa gaagaagaaa tcacagaccg tgcctaacag cgacagtcca
     3721 gatctgctga ccctgcaaaa gagcatccac tctgctagca ccccaagctc ccggcctaag
     3781 gagtggaggc ctgagaagat tagcgacctg cagacccagt cccagccact gaagtctctg
     3841 aggaagctgc tgcatctgtc aagtgcctcc aatcaccctg ctagcagcga ccccagattc
     3901 cagcccctga ccgcccagca gacaaagaat tccttcagcg agatcagaat ccaccccctg
     3961 agccaggcca gcggcgggtc ctccaatatc agacaggagc cagctcccaa aggccggcca
     4021 gccctgcagc tgcccggaca gatggacccc ggctggcacg tttcaagtgt gacaaggtcc
     4081 gccaccgagg gcccatccta ctccgaacag ctgggcgcca agagcgggcc caatgggcac
     4141 ccctataaca gaaccaaccg cagccggatg cccaacctga acgacctgaa ggagactgcc
     4201 ctgttggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     4261 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     4321 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     4381 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     4441 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     4501 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     4561 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4621 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     4681 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     4741 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     4801 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc
     4861 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     4921 ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca
     4981 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     5041 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     5101 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5161 gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta
     5221 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5281 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5341 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5401 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5461 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5521 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5581 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5641 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5701 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5761 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     5821 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5881 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5941 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     6001 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     6061 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     6121 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     6181 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     6241 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     6301 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     6361 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     6421 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     6481 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     6541 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     6601 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     6661 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     6721 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     6781 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     6841 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     6901 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     6961 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     7021 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     7081 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     7141 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     7201 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     7261 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     7321 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     7381 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     7441 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     7501 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7561 agatcaaagg atcttc
//
