LOCUS       sequence-442751-        7025 bp    DNA     circular SYN 15-APR-2025
DEFINITION  AAV expression of flag-mAdrb1-mNeonGreen from GfaABC1D promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7025)
  TITLE     Nagai Lab plasmids for expression in astrocytes and neurons
REFERENCE   2  (bases 1 to 7025)
  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"
COMMENT     Sequence Label: GfaABC1D-flag-mAdrb1-mNeonGreen-WPRE
FEATURES             Location/Qualifiers
     source          1..7025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     intron          1008..1140
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1123..1142
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1185..1203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1219..1243
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1276
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2684..2693
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2690..3400
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2945..2954
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3404..3428)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3438..4026
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3491..3511)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3909..3920)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4056..4073)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4062..4269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4277..4417
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4277..4406
                     /label=AAV2 ITR
     rep_origin      4492..4947
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4579..4598)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4789..4810
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4964..4983)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5083..5105
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5143..5161)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5229..5333
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5334..5402,5403..6194)
                     /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"
     rep_origin      6365..6953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6854..6873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7015..7025,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgtgt agttaatgat taacccgcca
      181 tgctacttat ctacgtagcc atgctctagg aagatctaac atatcctggt gtggagtagg
      241 ggacgctgct ctgacagagg ctcgggggcc tgagctggct ctgtgagctg gggaggaggc
      301 agacagccag gccttgtctg caagcagacc tggcagcatt gggctggccg ccccccaggg
      361 cctcctcttc atgcccagtg aatgactcac cttggcacag acacaatgtt cggggtgggc
      421 acagtgcctg cttcccgccg caccccagcc cccctcaaat gccttccgag aagcccattg
      481 agcagggggc ttgcattgca ccccagcctg acagcctggc atcttgggat aaaagcagca
      541 cagcccccta ggggctgccc ttgctgtgtg gcgccaccgg cggtggagaa caaggctcta
      601 ttcagcctgt gcccaggaaa ggggatcagg ggatgcccag gcatggacag tgggtggcag
      661 ggggggagag gagggctgtc tgcttcccag aagtccaagg acacaaatgg gtgaggggag
      721 agctctcccc atagctgggc tgcggcccaa ccccaccccc tcaggctatg ccagggggtg
      781 ttgccagggg cacccgggca tcgccagtct agcccactcc ttcataaagc cctcgcatcc
      841 caggagcgag cagagccaga gcaggttgga gaggagacgc atcacctccg ctgctcgcaa
      901 gctttattgc ggtagtttat cacagttaaa ttgctaacgc agtcagtgct tctgacacaa
      961 cagtctcgaa cttaagctgc agaagttggt cgtgaggcac tgggcaggta agtatcaagg
     1021 ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag agaagactct
     1081 tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag
     1141 gtgtccactc ccagttcaat tacagctctt aaggctagag tacttaatac gactcactat
     1201 aggctagcct cgagaattcc aagtttgtac aaaaaagcag gctgccacca tggactacaa
     1261 ggacgacgat gacaagggcg cgggggcgct cgccctgggc gcctccgaac cctgcaacct
     1321 gtcgtccgcc gcgccgctgc ccgacggtgc ggccaccgcg gcgcggctgc tggtgctcgc
     1381 gtcgcctccc gcctcgctgc tgcctccagc cagcgagggc tcagcgccgc tgtcgcagca
     1441 gtggaccgcg ggtatgggcc tactgctggc gctcatcgtt ctgctcatcg tggtgggtaa
     1501 cgtgctggtg atcgtggcca tcgccaagac cccgcggctg cagacgctca ccaacctctt
     1561 catcatgtcc ctggccagcg ctgatctggt catgggattg ctggtggtgc ctttcggggc
     1621 caccatcgtg gtgtggggcc gctgggagta cggctccttc ttctgcgagc tctggacttc
     1681 ggtagatgtg ctgtgtgtga cggccagcat tgagaccctg tgtgtcatcg ccctggaccg
     1741 ctacctcgcc atcacgtcgc cctttcgcta ccagagtttg ctgacgcgcg cgcgagcgcg
     1801 ggccctcgtg tgcacagtgt gggccatctc ggcgttggtg tccttcctgc ccatcctcat
     1861 gcactggtgg cgggccgaga gcgacgaagc gcgccgctgc tacaacgacc ccaagtgctg
     1921 cgatttcgtc accaacaggg cctacgccat cgcctcgtcc gtcgtctcct tctacgtgcc
     1981 cctgtgcatc atggccttcg tgtacctgcg ggtgttccgc gaggcccaaa aacaggtaaa
     2041 gaagatcgac agctgcgagc gccgcttcct cggcggccca gcccggccgc cctcgcctga
     2101 gccctcgccg tcacctgggc caccgcgccc cgcagactcg ctggccaacg ggcgctccag
     2161 caagcggcgg ccgtcgcgcc tcgtggctct gcgcgagcag aaggcgctca agacactggg
     2221 catcatcatg ggtgtgttca cgctctgctg gctgcccttc ttcctggcca acgtggtgaa
     2281 ggctttccac cgcgacctgg tgccggatcg cctcttcgtc ttcttcaact ggctgggcta
     2341 cgccaactcg gccttcaacc ccatcatcta ctgccgcagc cccgacttcc gcaaggcttt
     2401 ccagcgcctg ctctgctgcg cgcgccgggc cgcctgcaga cgccgcgcag cccacgggga
     2461 ccggccgcgc gcctccggct gcctggcgag agctgggccg ccgccgtccc ccggagctcc
     2521 ctcggacgac gacgacgacg acgccgggac caccccaccg gcgcgcctgc tggagccctg
     2581 gaccggctgc aacggcggga caaccactgt ggacagcgat tcgagcctgg acgagccggg
     2641 gcgccagggc ttctcctcgg agtccaaggt ggatccaccg gtcgccacca tggtgagcaa
     2701 gggcgaggag gataacatgg cctctctccc agcgacacat gagttacaca tctttggctc
     2761 catcaacggt gtggactttg acatggtggg tcagggcacc ggcaatccaa atgatggtta
     2821 tgaggagtta aacctgaagt ccaccaaggg tgacctccag ttctccccct ggattctggt
     2881 ccctcatatc gggtatggct tccatcagta cctgccctac cctgacggga tgtcgccttt
     2941 ccaggccgcc atggtagatg gctccggata ccaagtccat cgcacaatgc agtttgaaga
     3001 tggtgcctcc cttactgtta actaccgcta cacctacgag ggaagccaca tcaaaggaga
     3061 ggcccaggtg aaggggactg gtttccctgc tgacggtcct gtgatgacca actcgctgac
     3121 cgctgcggac tggtgcaggt cgaagaagac ttaccccaac gacaaaacca tcatcagtac
     3181 ctttaagtgg agttacacca ctggaaatgg caagcgctac cggagcactg cgcggaccac
     3241 ctacaccttt gccaagccaa tggcggctaa ctatctgaag aaccagccga tgtacgtgtt
     3301 ccgtaagacg gagctcaagc actccaagac cgagctcaac ttcaaggagt ggcaaaaggc
     3361 ctttaccgat gtgatgggca tggacgagct gtacaagtga tagacccagc tttcttgtac
     3421 aaagtgggaa ttccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3481 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3541 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3601 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3661 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3721 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3781 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     3841 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3901 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3961 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4021 ccccgcatcg ggaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     4081 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4141 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4201 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     4261 tgctggggag ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4321 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4381 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     4441 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     4501 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4561 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4621 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4681 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     4741 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4801 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     4861 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4921 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     4981 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5041 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5101 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5161 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5221 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5281 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5341 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5401 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5461 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5521 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5581 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5641 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5701 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5761 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5821 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5881 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5941 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6001 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     6061 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6121 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6181 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6241 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6301 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6361 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6421 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6481 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6541 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6601 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6661 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6721 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6781 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6841 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6901 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6961 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca
     7021 ggcag
//
