LOCUS       sequence-490429-        7636 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  Mammalian expression of CDKL5 isoform1 (1-960) L960D with N-terminal
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7636)
  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 7636)
  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: GFP-CDKL5(isoform1)_L960D
FEATURES             Location/Qualifiers
     source          1..7636
                     /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"
     CDS             1311..1355
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     regulatory      1371..1380
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1377..1379,1380..1382,1383..2093)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1377..1379,1380..1382,1383..2093)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1422..1443)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1683..1702)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2030..2051
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5127..5248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5164..5183)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5218..5237
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5255..5710)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5392..5413)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5604..5623
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5737..5841
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5843..6200
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5843..6034
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5866..5886)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6051..6186
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6235..7029
                     /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             6235..7029
                     /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(6289..6308)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6899..6918
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7217..7236)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7261..7308
                     /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 tccgctagac catggcttcc ggcctgaacg
     1321 acatcttcga ggctcagaaa atcgaatggc acgaaggagg cggaccggtc gccaccatgg
     1381 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1441 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1501 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1561 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1621 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1681 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1741 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1801 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1861 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1921 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1981 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2041 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac
     2101 tcagatctcg agacatgaag attcctaaca ttggtgatgt gatgaataaa tttgagatcc
     2161 ttggggttgt aggtgaagga gcctatggag ttgtacttaa atgcagacac aaggaaacac
     2221 atgaaattgt ggcgatcaag aaattcaagg acagtgaaga aaatgaagaa gtcaaagaaa
     2281 cgactttacg agagcttaaa atgcttcgga ctctcaagca ggaaaacatt gtggagttga
     2341 aggaagcatt tcgtcggagg ggaaagttgt acttggtgtt tgagtatgtt gaaaaaaata
     2401 tgctcgaatt gctggaagaa atgccaaatg gagttccacc tgagaaagta aaaagctaca
     2461 tctatcagct aatcaaggct attcactggt gccataagaa tgatattgtc catcgagata
     2521 taaaaccaga aaatctctta atcagccaca atgatgtcct aaaactgtgt gactttggtt
     2581 ttgctcgtaa tctgtcagaa ggcaataatg ctaattacac agagtacgtt gccaccagat
     2641 ggtatcggtc cccagaactc ttacttggcg ctccctatgg aaagtccgtg gacatgtggt
     2701 cggtgggctg tattcttggg gagcttagcg atggacagcc tttatttcct ggagaaagtg
     2761 aaattgacca actttttact attcagaagg tgctaggacc acttccatct gagcagatga
     2821 agcttttcta cagtaatcct cgcttccatg ggctccggtt tccagctgtt aaccatcctc
     2881 agtccttgga aagaagatac cttggaattt tgaatagtgt tctacttgac ctaatgaaga
     2941 atttactgaa gttggaccca gctgacagat acttgacaga acagtgtttg aatcacccta
     3001 catttcaaac ccagagactt ctggatcgtt ctccttcaag gtcagcaaaa agaaaacctt
     3061 accatgtgga aagcagcaca ttgtctaata gaaaccaagc cggcaaaagt actgctttgc
     3121 agtctcacca cagatctaac agcaaggaca tccagaacct gagtgtaggc ctgccccggg
     3181 ctgacgaagg tctccctgcc aatgaaagct tcctaaatgg aaaccttgct ggagctagtc
     3241 ttagtccact gcacaccaaa acctaccaag caagcagcca gcctgggtct accagcaaag
     3301 atctcaccaa caacaacata ccacaccttc ttagcccaaa agaagccaag tcaaaaacag
     3361 agtttgattt taatattgac ccaaagcctt cagaaggccc agggacaaag tacctcaagt
     3421 caaacagcag atctcagcag aaccgccact cattcatgga aagctctcaa agcaaagctg
     3481 ggacactgca gcccaatgaa aagcagagtc ggcatagcta tattgacaca attccccagt
     3541 cctctaggag tccctcctac aggaccaagg ccaaaagcca tggggcactg agtgactcca
     3601 agtctgtgag caacctttct gaagccaggg cccaaattgc ggagcccagt accagtaggt
     3661 acttcccatc tagctgctta gacttgaatt ctcccaccag cccaaccccc accagacaca
     3721 gtgacacgag aactttgctc agcccttctg gaagaaataa ccgaaatgag ggaacgctgg
     3781 actcacgtcg aaccacaacc agacattcta agacgatggg ggaattgaag ctgccggagc
     3841 acatggacag tagccattcc cattcactgt ctgcacctca cgaatctttt tcttatggac
     3901 tgggctacac cagccccttt tcttcccagc aacgtcctca taggcattct atgtatgtga
     3961 cccgtgacaa agtgagagcc aagggcttgg atggaagctt gagcataggg caagggatgg
     4021 cagctagagc caacagcctg caactcttgt caccccagcc tggagaacag ctccctccag
     4081 agatgactgt ggcaagatct tcggtcaaag agacctccag agaaggcacc tcttccttcc
     4141 atacacgcca gaagtctgag ggtggagtgt atcatgaccc acactctgat gatggcacag
     4201 cccccaaaga aaatagacac ctatacaatg atcctgtgcc aaggagagtt ggtagctttt
     4261 acagagtgcc atctccacgt ccagacaatt ctttccatga aaataatgtg tcaactagag
     4321 tttcttctct accatcagag agcagttctg gtaccaacca tagcaagcgg cagcctgcct
     4381 tcgacccctg gaagtcacct gagaacattt ctcatagcga gcagctgaag gaaaaggaaa
     4441 agcagggctt cttcaggagt atgaaaaaga agaagaagaa atcacagacc gtgcctaaca
     4501 gcgacagtcc agatctgctg accctgcaaa agagcatcca ctctgctagc accccaagct
     4561 cccggcctaa ggagtggagg cctgagaaga ttagcgacct gcagacccag tcccagccac
     4621 tgaagtctct gaggaagctg ctgcatctgt caagtgcctc caatcaccct gctagcagcg
     4681 accccagatt ccagcccctg accgcccagc agacaaagaa ttccttcagc gagatcagaa
     4741 tccaccccct gagccaggcc agcggcgggt cctccaatat cagacaggag ccagctccca
     4801 aaggccggcc agccctgcag ctgcccggac agatggaccc cggctggcac gtttcaagtg
     4861 tgacaaggtc cgccaccgag ggcccatcct actccgaaca gctgggcgcc aagagcgggc
     4921 ccaatgggca cccctataac agaaccaacc gcagccggat gcccaacctg aacgacctga
     4981 aggagactgc cgactaagga tccaccggat ctagataact gatcataatc agccatacca
     5041 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     5101 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     5161 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5221 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     5281 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5341 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5401 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5461 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5521 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5581 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5641 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5701 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5761 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5821 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     5881 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5941 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     6001 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     6061 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     6121 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     6181 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     6241 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     6301 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     6361 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     6421 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     6481 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     6541 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     6601 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     6661 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     6721 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     6781 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     6841 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     6901 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     6961 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     7021 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     7081 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     7141 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     7201 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     7261 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     7321 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     7381 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     7441 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     7501 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     7561 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7621 agatcaaagg atcttc
//
