LOCUS       sequence-82199-e        6344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express EGFP-Kir4.1 fusion protein in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6344)
  AUTHORS   Tong X, Ao Y, Faas GC, Nwaobi SE, Xu J, Haustein MD, Anderson MA, 
            Mody I, Olsen ML, Sofroniew MV, Khakh BS
  TITLE     Astrocyte Kir4.1 ion channel deficits contribute to neuronal 
            dysfunction in Huntington's disease model mice.
  JOURNAL   Nat Neurosci. 2014 May;17(5):694-703. doi: 10.1038/nn.3691. Epub 
            2014 Mar 30.
  PUBMED    24686787
REFERENCE   2  (bases 1 to 6344)
  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"; doi: "10.1038/nn.3691"; 
            journalName: "Nat Neurosci"; date: "2014-05"; volume: "17"; issue: 
            "5"; pages: "694-703"
COMMENT     Sequence Label: pZac2.1 gfaABC1D-EGFP-Kir4.1
FEATURES             Location/Qualifiers
     source          1..6344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1440..1449
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1446..1448,1449..1451,1452..2162)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1446..1448,1449..1451,1452..2162)
                     /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(1491..1512)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1752..1771)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2099..2120
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3369..3490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3380..3399)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3434..3453
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3652..3781
                     /label=AAV2 ITR
     primer_bind     complement(3800..3817)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3800..3816)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3809..3831)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3958..4413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4045..4064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4255..4276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4439..4543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4544..4612,4613..5404)
                     /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      5575..6163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6064..6083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcg
     1441 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1501 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1561 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1621 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1681 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1741 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1801 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1861 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1921 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1981 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2041 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2101 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2161 agtccggact cagatctcga gctcaagctt cgaattctgt tgccaaggtc tattacagcc
     2221 agacgacgca gacagagagc cggcccctag tggccccagg aatacgtcgg aggagggtcc
     2281 tgacaaaaga tggccggagc aacgtgagaa tggagcatat tgctgacaag cgtttcctct
     2341 acctcaagga tctatggacg accttcattg acatgcagtg gcgctacaag cttctgctct
     2401 tctcggcaac ctttgcaggc acttggttcc tctttggcgt ggtgtggtat ctggtcgctg
     2461 tggcccacgg ggacctgttg gagctgggac ctcctgccaa ccacacgccc tgtgtggtgc
     2521 aggtgcacac acttactggg gccttcctct tctccctcga atcccagacc accattggct
     2581 atggcttccg ctacatcagc gaggaatgcc ctctggccat tgtgcttctc attgcacagc
     2641 tcgtgctcac caccattctg gaaatcttca tcaccggaac cttccttgca aagattgccc
     2701 ggccgaagaa gagggctgag acgatccgtt tcagccagca tgcggttgtg gcttaccaca
     2761 acgggaagct ttgcctcatg atccgggtgg ccaacatgcg taagagtctc ctcattgggt
     2821 gccaggtgac aggcaaactg cttcaaaccc accagacaaa ggagggtgag aatattcggc
     2881 tcaaccaggt caatgtgact ttccaagtag acacagcctc tgatagcccc tttctcattc
     2941 tacccctgac tttctaccat gtggtagatg agaccagccc cttgaaagat ctccccctcc
     3001 gcagcgggga gggtgacttc gagctcgtgc tgatcctaag tgggacggtg gagtccacca
     3061 gcgccacctg tcaagttcgc acttcctatc taccggagga gatcctctgg ggctacgagt
     3121 tcacacctgc tatctcactg tcagccagtg gcaaatacgt ggctgacttc agcctttttg
     3181 accaggttgt gaaagtggcg tcccccggtg gtctccgaga tagcaccgta cgttatggag
     3241 acccagaaaa gctcaagttg gaggagtcat taagagagca agctgaaaag gaaggcagtg
     3301 cccttagtgt gcgcattagt aacgtctgat ctagagtcga cccgggcggc cgcttcgagc
     3361 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3421 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3481 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     3541 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatcttcc
     3601 tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc
     3661 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3721 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3781 gccttaatta acctaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     3841 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     3901 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     3961 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4021 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4081 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4141 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4201 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4261 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4321 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4381 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     4441 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4501 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4561 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4621 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4681 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4741 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4801 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4861 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4921 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4981 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5041 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5101 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5161 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5221 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5281 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5341 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5401 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5461 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5521 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5581 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5641 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5701 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5761 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5821 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5881 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5941 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6001 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6061 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6241 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6301 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
