LOCUS       sequence-483102-        5754 bp    DNA     circular SYN 13-AUG-2025
DEFINITION  FRT-dependent expression of fluorescent protein, mNeonGreen, in 
            astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5754)
  TITLE     Nagai Lab plasmids for expression in astrocytes and neurons
REFERENCE   2  (bases 1 to 5754)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 13, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: GfaABC1D-fDIO-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..5754
                     /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"
     protein_bind    1250..1283
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(1376..2086)
                     /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      1822..1831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2083..2092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2099..2132)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(2133..2157)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2167..2755
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2220..2240)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2638..2649)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2785..2802)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2791..2998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3006..3146
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3006..3135
                     /label=AAV2 ITR
     rep_origin      3221..3676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3308..3327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3518..3539
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3693..3712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3812..3834
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3872..3890)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3958..4062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4063..4131,4132..4923)
                     /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      5094..5682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5583..5602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5744..5754,1..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 gctgccaccg aagttcctat
     1261 tctctagaaa gtataggaac ttcgcagaat ggtagctgga ttgtagctgc tattagcaat
     1321 atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcggc gcgcctcact
     1381 tgtacagctc gtccatgccc atcacatcgg taaaggcctt ttgccactcc ttgaagttga
     1441 gctcggtctt ggagtgcttg agctccgtct tacggaacac gtacatcggc tggttcttca
     1501 gatagttagc cgccattggc ttggcaaagg tgtaggtggt ccgcgcagtg ctccggtagc
     1561 gcttgccatt tccagtggtg taactccact taaaggtact gatgatggtt ttgtcgttgg
     1621 ggtaagtctt cttcgacctg caccagtccg cagcggtcag cgagttggtc atcacaggac
     1681 cgtcagcagg gaaaccagtc cccttcacct gggcctctcc tttgatgtgg cttccctcgt
     1741 aggtgtagcg gtagttaaca gtaagggagg caccatcttc aaactgcatt gtgcgatgga
     1801 cttggtatcc ggagccatct accatggcgg cctggaaagg cgacatcccg tcagggtagg
     1861 gcaggtactg atggaagcca tacccgatat gagggaccag aatccagggg gagaactgga
     1921 ggtcaccctt ggtggacttc aggtttaact cctcataacc atcatttgga ttgccggtgc
     1981 cctgacccac catgtcaaag tccacaccgt tgatggagcc aaagatgtgt aactcatgtg
     2041 tcgctgggag agaggccatg ttatcctcct cgcccttgct caccatggtg gcgctagcga
     2101 agttcctata ctttctagag aataggaact tcacccagct ttcttgtaca aagtgggaat
     2161 tccgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2221 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2281 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2341 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     2401 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     2461 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     2521 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg
     2581 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     2641 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     2701 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcgg
     2761 gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2821 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2881 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2941 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagg
     3001 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3061 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3121 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3181 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3241 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     3301 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3361 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3421 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3481 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3541 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     3601 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3661 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3721 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3781 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3841 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3901 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3961 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4021 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4081 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4141 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4201 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4261 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4321 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4381 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4441 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4501 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4561 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4621 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4681 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4741 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     4801 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4861 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4921 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4981 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5041 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5101 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5161 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5221 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5281 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5341 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5401 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5461 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5521 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5581 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5641 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5701 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcag
//
