LOCUS       sequence-344763-        5778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SPN-ARR fragment with structure opening N52R and actin binding site 
            Q37A/R38A mutations in EGFP backbone for mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5778)
  AUTHORS   Salomaa SI, Miihkinen M, Kremneva E, Paatero I, Lilja J, Jacquemet 
            G, Vuorio J, Antenucci L, Kogan K, Hassani Nia F, Hollos P, Isomursu
            A, Vattulainen I, Coffey ET, Kreienkamp HJ, Lappalainen P, Ivaska J
  TITLE     SHANK3 conformation regulates direct actin binding and crosstalk 
            with Rap1 signaling.
  JOURNAL   Curr Biol. 2021 Sep 29. pii: S0960-9822(21)01262-8. doi: 
            10.1016/j.cub.2021.09.022.
  PUBMED    34610274
REFERENCE   2  (bases 1 to 5778)
  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: "Curr Biol. 
            2021 Sep 29. pii: S0960-9822(21)01262-8. doi: 
            10.1016/j.cub.2021.09.022."
COMMENT     Sequence Label: EGFP-SPN_ARR Q37A/R38A/N52R (Shank3)
FEATURES             Location/Qualifiers
     source          1..5778
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2566..2687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2603..2622)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2657..2676
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2694..3149)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2831..2852)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3043..3062
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3176..3280
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3282..3639
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3282..3473
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3305..3325)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3490..3625
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3674..4468
                     /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             3674..4468
                     /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(3728..3747)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4338..4357
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4656..4675)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4700..4747
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5076..5664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5565..5584
                     /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 gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attctgggtc gaccatggac
     1381 ggccccgggg ccagcgccgt ggtcgtgcgc gtcggcatcc cggacctgca acaaacgaag
     1441 tgcctgcgtc tggatccaac cgcgcccgtg tgggccgcca aggctgctgt gctctgcgcc
     1501 ctcaaccaca gccttcagga cgcgctcaga tacgggctat tccagcctcc ctcccggggt
     1561 cgcgccggca agttcctgga tgaagagcgg ctcttacagg actacccgcc taacctggac
     1621 acgcccctgc cctatctgga gtttcgatac aagcggagag tttatgccca gaacctcata
     1681 gatgacaagc agtttgcaaa gctgcacaca aaggcaaacc tgaagaagtt catggactat
     1741 gtccagctac acagcacaga caaggtcgcc cgcctgctgg acaaggggct ggaccccaat
     1801 ttccatgacc ctgactcagg agagtgccct ctgagccttg cagcacagtt ggacaacgcc
     1861 aatgacctcc tgaaggttct tcgcaatggc ggtgctcatc tggacttccg aacccgggat
     1921 gggctaaccg ctgtccactg cgccacgcga cagcggaatg cgggagcatt gacgaccctg
     1981 ctggacctgg gcgcttcacc tgactacaag gacagccgcg gcctgacgcc cctgtaccat
     2041 agtgccctag gcggcgggga tgccctctgc tgtgagctgc ttctccatga tcacgcacag
     2101 ttggggacca ctgacgagaa tggctggcag gagatccatc aggcctgtcg ctttgggcat
     2161 gtacagcact tggagcacct gctgttctat ggggccaaca tgggtgccca gaacgcctcc
     2221 ggaaacacag ccttgcacat ctgtgccctc tataaccagg agagctgtgc ccgcgtcctg
     2281 cttttccgtg gtgccaacaa ggacgtccgc aattacaaca gccagacagc cttccaggtc
     2341 gccattattg cagggaactt tgagcttgcg gaggtaatca agacccacaa agactcggat
     2401 gtcgtaccat tcagggaata aggtaccgcg ggcccgggat ccaccggatc tagataactg
     2461 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2521 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     2581 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2641 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     2701 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2761 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2821 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2881 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2941 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3001 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3061 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3121 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     3181 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3241 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     3301 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3361 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3481 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3541 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     3601 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     3661 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3721 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3781 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3841 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3901 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3961 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4021 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4081 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4141 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4201 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4261 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4321 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4381 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4441 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4501 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4561 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4621 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     4681 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     4741 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     4801 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     4861 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     4921 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4981 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5041 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5101 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5161 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5221 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5281 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5341 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5401 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5461 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5521 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5581 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5641 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5701 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5761 cgtattaccg ccatgcat
//
