LOCUS       sequence-490451-        6045 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  Bacterial expression of SHANK3 PDZ domain (570-664).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6045)
  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 6045)
  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: mSHANK3_PDZ
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(746..764)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      760..807
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1045..1063)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1131..1235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1236..1304,1305..2096)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1454..1473)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2267..2855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2756..2775
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3009..3026
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3041..3180
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3106..3125)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3266..3288
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3282..3473)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4785..5867)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5829..5848
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5868..5945)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga attgatccct tcaccgttgc aatcctgcag aaacgtgatc
      421 atgaaggttt tggttttgtt ctgcgtggtg caaaagcaga aaccccgatt gaagaattta
      481 ccccgacacc ggcatttccg gcactgcagt atctggaaag cgttgatgtt gaaggtgttg
      541 catggcgtgc aggtctgcgt accggtgatt ttctgattga agttaatggt gtgaacgtgg
      601 tgaaagtggg tcataaacag gttgttggtc tgattcgtca aggtggtaat cgtctggtta
      661 tgaaagttgt tagcgtgacc taaaagggcg agctcagatc cggctgctaa caaagcccga
      721 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      781 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca
      841 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
      901 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
      961 ttaactgtga taaactaccg cattaaagct agcttatcga tgataagctg tcaaacatga
     1021 gaattaattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     1081 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1141 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1201 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1261 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1321 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1381 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1441 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     1501 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1561 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1621 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1681 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1741 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     1801 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1861 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1921 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1981 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2041 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2101 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2161 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2221 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2281 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2341 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2401 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2461 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2521 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2581 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2641 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2701 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2761 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2821 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2881 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2941 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3001 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3061 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3121 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3181 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3241 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3301 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3361 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3421 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3481 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3541 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3601 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3661 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3721 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3781 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3841 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     3901 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc
     3961 gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg
     4021 ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg
     4081 aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac
     4141 cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc catgccaacc
     4201 cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg
     4261 aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta
     4321 cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa
     4381 tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg
     4441 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4501 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4561 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4621 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4681 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4741 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4801 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4861 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4921 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4981 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5041 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     5101 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5161 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5221 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5281 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5341 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5401 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5461 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5521 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5581 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5641 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5701 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5761 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5821 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5881 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5941 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     6001 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatg
//
