LOCUS       sequence-442754-        6250 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cre-dependently expresses mNeonGreen in astorocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6250)
  TITLE     Nagai Lab plasmids for expression in astrocytes and neurons
REFERENCE   2  (bases 1 to 6250)
  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-DIO-mNeonGreen-WPRE
FEATURES             Location/Qualifiers
     source          1..6250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    262..283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(298..303,304..321,322..328)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    336..352
                     /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     341..363
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     360..376
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     360..376
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   414..543
                     /label=AAV2 ITR
     intron          1394..1526
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1509..1528
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1571..1589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1644..1663
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1698..1731
                     /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    1794..1827
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1851..2561)
                     /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      2297..2306
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2558..2567
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2584..2617)
                     /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(2688..2721)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2813..3401
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2866..2886)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3284..3295)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3430..3551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3441..3460)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3495..3514
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3713..3842
                     /label=AAV2 ITR
     primer_bind     complement(3861..3878)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3861..3877)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3870..3892)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4019..4474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4106..4125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4316..4337
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4500..4604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4605..4673,4674..5465)
                     /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      5636..6224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6125..6144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
       61 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      121 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
      181 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
      241 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      301 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      361 aggaaacagc tatgaccatg attacgccag atttaattaa ggccttaatt aggctgcgcg
      421 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
      481 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
      541 ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga
      601 tctaacatat cctggtgtgg agtaggggac gctgctctga cagaggctcg ggggcctgag
      661 ctggctctgt gagctgggga ggaggcagac agccaggcct tgtctgcaag cagacctggc
      721 agcattgggc tggccgcccc ccagggcctc ctcttcatgc ccagtgaatg actcaccttg
      781 gcacagacac aatgttcggg gtgggcacag tgcctgcttc ccgccgcacc ccagcccccc
      841 tcaaatgcct tccgagaagc ccattgagca gggggcttgc attgcacccc agcctgacag
      901 cctggcatct tgggataaaa gcagcacagc cccctagggg ctgcccttgc tgtgtggcgc
      961 caccggcggt ggagaacaag gctctattca gcctgtgccc aggaaagggg atcaggggat
     1021 gcccaggcat ggacagtggg tggcaggggg ggagaggagg gctgtctgct tcccagaagt
     1081 ccaaggacac aaatgggtga ggggagagct ctccccatag ctgggctgcg gcccaacccc
     1141 accccctcag gctatgccag ggggtgttgc caggggcacc cgggcatcgc cagtctagcc
     1201 cactccttca taaagccctc gcatcccagg agcgagcaga gccagagcag gttggagagg
     1261 agacgcatca cctccgctgc tcgcaagctt tattgcggta gtttatcaca gttaaattgc
     1321 taacgcagtc agtgcttctg acacaacagt ctcgaactta agctgcagaa gttggtcgtg
     1381 aggcactggg caggtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact
     1441 gggcttgtcg agacagagaa gactcttgcg tttctgatag gcacctattg gtcttactga
     1501 catccacttt gcctttctct ccacaggtgt ccactcccag ttcaattaca gctcttaagg
     1561 ctagagtact taatacgact cactataggc tagagcctct gctaaccatg ttcatgcctt
     1621 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     1681 caaagaattg gatccccata acttcgtata aagtatccta tacgaagtta tatcaaaata
     1741 ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cccataactt
     1801 cgtataatgt atgctatacg aagttatact agggatcccc cctcgagttt tcacttgtac
     1861 agctcgtcca tgcccatcac atcggtaaag gccttttgcc actccttgaa gttgagctcg
     1921 gtcttggagt gcttgagctc cgtcttacgg aacacgtaca tcggctggtt cttcagatag
     1981 ttagccgcca ttggcttggc aaaggtgtag gtggtccgcg cagtgctccg gtagcgcttg
     2041 ccatttccag tggtgtaact ccacttaaag gtactgatga tggttttgtc gttggggtaa
     2101 gtcttcttcg acctgcacca gtccgcagcg gtcagcgagt tggtcatcac aggaccgtca
     2161 gcagggaaac cagtcccctt cacctgggcc tctcctttga tgtggcttcc ctcgtaggtg
     2221 tagcggtagt taacagtaag ggaggcacca tcttcaaact gcattgtgcg atggacttgg
     2281 tatccggagc catctaccat ggcggcctgg aaaggcgaca tcccgtcagg gtagggcagg
     2341 tactgatgga agccataccc gatatgaggg accagaatcc agggggagaa ctggaggtca
     2401 cccttggtgg acttcaggtt taactcctca taaccatcat ttggattgcc ggtgccctga
     2461 cccaccatgt caaagtccac accgttgatg gagccaaaga tgtgtaactc atgtgtcgct
     2521 gggagagagg ccatgttatc ctcctcgccc ttgctcacca tggtggcggt accgaattct
     2581 agtataactt cgtataggat actttatacg aagttatcat tgggattctt cctattttga
     2641 tccaagcatc accatcgacc ctctagtcca gatctcacca tcgacccata acttcgtata
     2701 gcatacatta tacgaagtta tgtccctcga agaggttcga attcgatatc aagcttctgc
     2761 agtcgacgct agcgggccgc gactctagat cataatcagc cataccacgc ataatcaacc
     2821 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     2881 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     2941 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3001 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3061 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3121 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3181 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     3241 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3301 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3361 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcggccgg ccgcttcgag
     3421 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3481 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3541 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     3601 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc
     3661 ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     3721 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3781 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3841 agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3901 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3961 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4021 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4081 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4141 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4201 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4261 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4321 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4381 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4441 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4741 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4801 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4861 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4921 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4981 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5041 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5101 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5161 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5221 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5281 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5341 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5401 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5461 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5521 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5581 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5761 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5821 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5881 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5941 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6001 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6061 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6121 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6181 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6241 cctttttacg
//
