LOCUS       sequence-313797-        7388 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Conditionally overexpress collybistin (CB2SH3-) and mCitrine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7388)
  AUTHORS   George S, James S, de Blas AL
  TITLE     Selective overexpression of Collybistin in mouse hippocampal 
            pyramidal cells enhances GABAergic neurotransmission and protects 
            against PTZ-induced seizures.
  JOURNAL   eNeuro. 2021 Jun 2. pii: ENEURO.0561-20.2021. doi: 
            10.1523/ENEURO.0561-20.2021.
  PUBMED    34083383
REFERENCE   2  (bases 1 to 7388)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eNeuro. 
            2021 Jun 2. pii: ENEURO.0561-20.2021. doi: 
            10.1523/ENEURO.0561-20.2021."
COMMENT     Sequence Label: pAAV-hSyn-DIO-HA-CB2SH3(-)-IRES-mCitrine
FEATURES             Location/Qualifiers
     source          1..7388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     70..87
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(1499..1525)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1539..1572
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1623..1656
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1681..2269
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1734..1754)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2152..2163)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2301..2777
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2500..2519)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2817..2957
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2817..2946
                     /label=AAV2 ITR
     rep_origin      3032..3487
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3119..3138)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3329..3350
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3504..3523)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3623..3645
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3683..3701)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3769..3873
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3874..3942,3943..4734)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4092..4111)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4905..5493
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5394..5413
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5555..5684
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        5729..6176
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(6214..6247)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(6298..6331)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(6346..7056,7057..7059,7060..7062))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6388..6409)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6737..6756
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6996..7017
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      7059..7068
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(7069..7388,1..254)
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7229..7248)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
ORIGIN
        1 ctacagacgt tgtttgtctt caagaagctt ccagaggaac tgcttccttc acgacattca
       61 acagaccttg cattcctttg gcgagagggg aaagacccct aggaatgctc gtcaagaaga
      121 cagggccagg tttccgggcc ctcacattgc caaaagacgg caatatggtg gaaaataaca
      181 tatagacaaa cgcacaccgg ccttattcca agcggcttcg gccagtaacg ttaggggggg
      241 gggagggaga ggggcgatat cactaatagt gccattttct ttgggttact ttctgtttag
      301 acgcttttct cacagtcatt gcagcctgtc tcttctgatt ttcagaaatc tcaaagccaa
      361 ttttttcatc ttcttgtacc attttcctct cttctctgaa agctctaagc caacgtattt
      421 tctcctccag tttcttggca aagaacagat gtacttcttc ggtttccttg ttgtgcagct
      481 tgaaagcatt tttcatgctg acattaaagt catcatctct gccatcttca atgtcaatga
      541 cctcatattt atccatgtca atgcgcccct tgtagtatag gatgtctctc cggattaggt
      601 ccttcttgca gaggaccatc tggtggtcaa acaggaagaa gactctctgc tggttgcggc
      661 catagggctg gtagatccag gccatctctc cagtgtagat cagttccgag ctcctgtcta
      721 agatgtcatc tccctcccag tctaggacag aggcctgcca ttgagcaatc ttgtcaatat
      781 tctccagacg tcgcttgcgt tcattgatct gctgagtcac atttctcatg acagccaaag
      841 cagctgcgac atatctgtaa tcactgtggt cttgggcagt atactttagg agctcggcca
      901 attgtaaggg gtatttgcag atcttctgta ctggagtcaa aagaaaacca tcgatagcga
      961 tgtcgatcat ctgctgcaag aggcggcagg cctcaaagaa gtgttgatag cggctgtcct
     1021 tcatcagttt ggagagctcc atgcaggcat ccaggtggtt gttacaatac tcagagtata
     1081 tccagaagcc atcttgatgc tccaagaagc agggtcctat ctcgctgagg tgggggtcat
     1141 cattgttgta ctgcttctcc aagtctctca cgaagcccat ctgaaatctg tagatgtctt
     1201 caatgtttcc aaagatgact ttcagctgct catcactgaa catgtccctt ctctttcggc
     1261 actgcttcag gtacccctcg caaatgtcct tgagatgctt gatgtaatga cgttccgtgc
     1321 tcatgatctc attgatgaca ttggcccgca tctggtcccg gttctgtagt ggccggccca
     1381 gacagaggca gtctgagttg gggtccaggt gcccattctg cacgtcactg ggcccttcct
     1441 ccaccccatc ctcctggttc acccagagca ttcccgatcc gcctctaatc cactgcatag
     1501 cgtaatctgg aacatcgtat gggtacatgg tggctagcat aacttcgtat aaagtatcct
     1561 atacgaagtt atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa
     1621 gaataacttc gtataatgta tgctatacga agttatgaat tcgatatcaa gcttatcgat
     1681 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     1741 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     1801 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     1861 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     1921 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     1981 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     2041 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     2101 gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     2161 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     2221 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga
     2281 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     2341 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     2401 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     2461 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     2521 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     2581 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     2641 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     2701 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     2761 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     2821 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     2881 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     2941 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3001 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     3061 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     3121 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3181 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3241 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3301 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3361 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     3421 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     3481 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3541 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3601 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3661 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3721 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3781 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3841 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3901 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3961 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4021 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4081 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4141 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4201 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4261 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4321 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4381 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4441 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4501 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4561 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4621 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4681 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4741 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4801 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4861 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4921 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4981 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5041 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5101 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5161 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5221 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5281 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5341 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5401 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5461 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5521 ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg
     5581 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     5641 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtgt
     5701 gtctagactg cagagggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc
     5761 ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca
     5821 ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg
     5881 cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
     5941 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc
     6001 cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg
     6061 cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
     6121 cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga
     6181 gaaggtaccg gatcctctag agtcgactcc ggaataactt cgtataggat actttatacg
     6241 aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa cctcttaata
     6301 acttcgtata gcatacatta tacgaagtta tggcgcgccc tatcacttgt acagctcgtc
     6361 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     6421 ctcgttgggg tctttgctca gtttggactg gtagctcagg tagtggttgt cgggcagcag
     6481 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     6541 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     6601 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     6661 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     6721 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     6781 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     6841 gcgggcgaag cacatcaggc cgtagccgaa ggtggtcacg agggtgggcc agggcacggg
     6901 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     6961 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     7021 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggctt atcatcgtgt
     7081 ttttcaaagg aaaaccacgt ccccgtggtt cggggggcct agacgttttt ttaacctcga
     7141 ctaaacacat gtaaagcatg tgcaccgagg ccccagatca gatcccatac aatggggtac
     7201 cttctgggca tccttcagcc ccttgttgaa tacgcttgag gagagccatt tgactctttc
     7261 cacaactatc caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta
     7321 tacacgtggc ttttggccgc agaggcacct gtcgccaggt ggggggttcc gctgcctgca
     7381 aagggtcg
//
