LOCUS       sequence-442756-        6976 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cre-dependently expresses mNeonGreen, mScarlet-I in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6976)
  TITLE     Nagai Lab plasmids for expression in astrocytes and neurons
REFERENCE   2  (bases 1 to 6976)
  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-mScarlet-l-WPRE
FEATURES             Location/Qualifiers
     source          1..6976
                     /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)."
     regulatory      2793..2802
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2799..3494
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2932..2950)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3200..3219
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3539..4127
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3592..3612)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4010..4021)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4156..4277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4167..4186)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4221..4240
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4439..4568
                     /label=AAV2 ITR
     primer_bind     complement(4587..4604)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4587..4603)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4596..4618)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4745..5200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4832..4851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5042..5063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5226..5330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5331..5399,5400..6191)
                     /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      6362..6950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6851..6870
                     /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 agcgggccca ctagtaccgg tcgccaccat ggtgagcaag ggcgaggcag
     2821 tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt
     2881 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     2941 aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt
     3001 acggctccag ggccttcatc aagcaccccg ccgacatccc cgactactat aagcagtcct
     3061 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg
     3121 tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca
     3181 ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca
     3241 ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc
     3301 tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg
     3361 tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg
     3421 actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg
     3481 acgagctgta caagtaaagc ggccgcgact ctagatcata atcagccata ccacgcataa
     3541 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3601 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3661 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3721 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3781 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3841 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3901 gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc
     3961 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4021 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4081 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc ggccggccgc
     4141 ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     4201 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     4261 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     4321 agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg
     4381 atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag
     4441 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4501 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4561 gcgcgcagcc ttaattaacc taattcactg gccgtcgttt tacaacgtcg tgactgggaa
     4621 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4681 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4741 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4801 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5041 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5101 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5161 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     5221 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5281 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5341 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5401 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5461 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5521 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5581 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5641 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5701 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5761 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5821 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5881 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5941 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6001 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6061 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6121 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6181 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6241 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6901 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6961 acgcggcctt tttacg
//
