LOCUS       sequence-491146-        7846 bp    DNA     circular SYN 30-OCT-2025
DEFINITION  Mammalian expression of CDKL5 isoform2 (1-1030) with N-terminal GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7846)
  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 7846)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 30, 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(isoform-2)
FEATURES             Location/Qualifiers
     source          1..7846
                     /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    5337..5458
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5374..5393)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5428..5447
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5465..5920)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5602..5623)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5814..5833
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5947..6051
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6053..6410
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6053..6244
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6076..6096)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6261..6396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6445..7239
                     /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             6445..7239
                     /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(6499..6518)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7109..7128
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7427..7446)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7471..7518
                     /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 ccactaggag 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 gaaccaacca ctcaaaaaga caaccagcat
     4381 tcgatccatg gaaaagtcct gaaaatatta gtcattcaga gcaactcaag gaaaaagaga
     4441 agcaaggatt tttcaggtca atgaaaaaga aaaagaagaa atctcaaaca gtacccaatt
     4501 ccgacagccc tgatcttctg acgttgcaga aatccattca ttctgctagc actccaagca
     4561 gcagaccaaa ggagtggcgc cccgagaaga tctcagatct gcagacccaa agccagccat
     4621 taaaatcact gcgcaagttg ttacatctct cttcggcctc aaatcacccg gcttcctcag
     4681 atccccgctt ccagccctta acagctcaac aaaccaaaaa ttccttctca gaaattcgga
     4741 ttcaccccct gagccaggcc tctggcggga gcagcaacat ccggcaggaa cccgcaccga
     4801 agggcaggcc agccctccag ctgccagacg gtggatgtga tggcagaaga cagagacacc
     4861 attctggacc ccaagataga cgcttcatgt taaggacgac agaacaacaa ggagaatact
     4921 tctgctgtgg tgacccaaag aagcctcaca ctccgtgcgt cccaaaccga gcccttcatc
     4981 gtccaatctc cagtcctgct ccctatccag tactccaggt ccgaggcact tccatgtgcc
     5041 cgacactcca ggtccgaggc actgatgctt tcagctgccc aacccagcaa tccgggttct
     5101 ctttcttcgt gagacacgtt atgagggaag ccctgattca cagggcccag gtaaaccaag
     5161 ctgcgctcct gacataccat gagaatgcgg cactgacggg caagtaagga tccaccggat
     5221 ctagataact gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     5281 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     5341 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5401 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac
     5461 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5521 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5581 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5641 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5701 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5761 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5821 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5881 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     5941 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6001 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     6061 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     6121 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     6181 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     6241 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     6301 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     6361 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     6421 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     6481 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     6541 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     6601 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     6661 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     6721 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     6781 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     6841 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     6901 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     6961 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     7021 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     7081 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     7141 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     7201 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     7261 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     7321 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     7381 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     7441 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     7501 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     7561 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     7621 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     7681 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7741 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7801 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
