LOCUS       sequence-258719-        7100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  conditional expression of a diphtheria toxin receptor (DTR)–GFP 
            fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7100)
  AUTHORS   Azim E, Jiang J, Alstermark B, Jessell TM
  TITLE     Skilled reaching relies on a V2a propriospinal internal copy 
            circuit.
  JOURNAL   Nature. 2014 Apr 17;508(7496):357-63. doi: 10.1038/nature13021. Epub
            2014 Feb 2.
  PUBMED    24487617
REFERENCE   2  (bases 1 to 7100)
  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/nature13021"; journalName: "Nature"; date: "2014-04-17- 
            17"; volume: "508"; issue: "7496"; pages: "357-63"
COMMENT     Sequence Label: pAAV-FLEX-DTR-GFP
FEATURES             Location/Qualifiers
     source          1..7100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    205..429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     418..435
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     misc_feature    462..1050
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             568..579
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     977..997
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1089..1122
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1193..1226
                     /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)."
     primer_bind     1253..1269
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1253..1269
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      1934..1943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1940..1942,1943..1945,1946..2659)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1940..1942,1943..1945,1946..2656)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1985..2006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2246..2265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2593..2614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2688..2704)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2711..2744)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2807..2840)
                     /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)."
     primer_bind     complement(2875..2894)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2956..2978)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3257..3534)
                     /label=chicken beta-actin promoter
     repeat_region   3840..3969
                     /label=AAV2 ITR
     promoter        complement(3990..4008)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3991..4008)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4298..4317)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(4531..4550)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4650..4672
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4710..4728)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4796..4900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4901..4969,4970..5761)
                     /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"
     primer_bind     complement(5119..5138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5932..6520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6421..6440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6761..6781)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6766..7095
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6946..7081
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc
       61 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt
      121 gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta gtcgagcccc
      181 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
      241 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
      301 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
      361 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
      421 aaggcacagt cgaggctgat cagcgagtcg acggtatcga tgcggggagg cggcccaaag
      481 ggagatccga ctcgtctgag ggcgaaggcg aagacgcgga agaggccgca gagccggcag
      541 caggccgcgg gaaggaaggt ccgctggatt gagggccgaa gggacgtagc agaaggacgt
      601 cccgcgcaga atccaggtgg caacacaggc gagcagccaa ggaaaggacg atgatttccc
      661 cgacaacacc acggaattgt cagtgcccaa cagccgagcc cctgtccagc agcgggcaag
      721 gcaggcggcg atgagttccg ccgtggcaat agggaggggg aaagcgaaag tcccggaaag
      781 gagctgacag gtggtggcaa tgccccaacc agtgggggtt gcgtcagcaa acacagtgca
      841 caccacgcca cgttgcctga caacgggcca caactcctca taaagagaca gcaaccagga
      901 tttatacaag gaggagaaaa tgaaagccat acgggaagca atagcatgat acaaaggcat
      961 taaagcagcg tatccacata gcgtaaaagg agcaacatag ttaagaatac cagtcaatct
     1021 ttcacaaatt ttgtaatcca gaggttgatt atcgataagc tgttaaagct tgaacctctt
     1081 cgagggacat aacttcgtat aatgtatgct atacgaagtt atgggtcgat ggtgagatct
     1141 ggactagagg gtcgatggtg atgcttggat caaaatagga agaatcccaa tgataacttc
     1201 gtataaagta tcctatacga agttatgata tcagatctct cgaggcggcc gctctagaac
     1261 tagtggatcc cccgggctgc aggaattcgc caccatgaag ctgctgccgt cggtggtgct
     1321 gaagctcctt ctggctgcag ttctttcggc actggtgact ggcgagagcc tggagcagct
     1381 tcggagaggg ctagctgctg gaaccagcaa cccggaccct tccactggat ctacggacca
     1441 gctgctacgc ctaggaggcg gccgggaccg gaaagtccgt gacttgcaag aggcagatct
     1501 ggaccttttg agagtcactt tatcctccaa gccacaagca ctggccacac caagcaagga
     1561 ggagcacggg aaaagaaaga agaaaggcaa gggactaggg aagaagaggg acccatgtct
     1621 tcggaaatac aaggacttct gcatccacgg agaatgcaaa tatgtgaagg agctccgggc
     1681 tccctcctgc atctgccacc caggttacca tggagagagg tgtcatgggc tgagcctccc
     1741 agtggaaaat cgcttatata cctatgacca tacaactatc ctggctgtgg tggccgtggt
     1801 gctgtcctct gtctgtctgc tggtcatcgt ggggcttctc atgtttaggt accataggag
     1861 aggtggttat gatgtggaaa acgaagagaa agtgaagttg ggcatgacta attcccaccc
     1921 ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1981 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2041 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2101 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2161 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2221 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2281 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2341 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2401 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2461 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2521 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2581 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2641 ggacgagctg tacaagtaaa gcggccggaa ttcgatatca agcttatcga taccgtcgac
     2701 ctcgaaattc ataacttcgt atagcataca ttatacgaag ttatgggtcg atggtgatgc
     2761 ttggcaattc gggtcgatgg tgaagcattg gtcttcctat tttgatataa cttcgtatag
     2821 gatactttat acgaagttat ggggatccaa ttctttgcca aaatgatgag acagcacaat
     2881 aaccagcacg ttgcccagga gctgtaggaa aaagaagaag gcatgaacat ggttagcaga
     2941 ggctctagag ccgccggtca cacgccagaa gccgaacccc gccctgcccc gtcccccccg
     3001 aaggcagccg tccccccgcg gacagccccg agccgctccc cccgcacaaa gggccctccc
     3061 ggagcccctc aaggctttca cgcagccaca gaaaagaaac aagccgtcat taaaccaagc
     3121 gctaattaca gcccggagga gaagggccgt cccgcccgct cacctgtggg agtaacgcgg
     3181 tcagtcagag ccggggcggg cggcgcgagg cggcggcgga gcggggcacg gggcgaaggc
     3241 agcgcgcagc gactcccgcc cgccgcgcgc ttcgcttttt atagggccgc cgccgccgcc
     3301 gcctcgccat aaaaggaaac tttcggagcg cgccgctctg attggctgcc gccgcacctc
     3361 yccgccycgc cccccccccc cccccccccc cccccccccc ccccggcgcg cgcccccccc
     3421 ccccccccgc ccccatcgct gcacaaaata attaaaaaat aaataaatac aaaattgggg
     3481 gtggggaggg gggggagatg gggagagtga agcagaacgt ggggctcacc tcgaccatgg
     3541 taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt
     3601 actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg
     3661 gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt
     3721 gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca
     3781 atggtaccta gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa
     3841 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     3901 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     3961 agcgcgcagc tagaattaat tccgtgtatt ctatagtgtc acctaaatcg tatgtgtatg
     4021 atacataagg ttatgtatta attgtagccg cgttctaacg acaatatgta caagcctaat
     4081 tgtgtagcat ctggcttact gaagcagacc ctatcatctc tctcgtaaac tgccgtcaga
     4141 gtcggtttgg ttggacgaac cttctgagtt tctggtaacg ccgtcccgca cccggaaatg
     4201 gtcagcgaac caatcagcag ggtcatcgct agccagatcc tctacgccgg acgcatcgtg
     4261 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     4321 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     4381 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     4441 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     4501 aagggagagc gtcgaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4561 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4621 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4681 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4741 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4801 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4861 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4921 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4981 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5041 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5101 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5161 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5221 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5281 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5341 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5401 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5461 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5521 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5581 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5641 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5701 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5761 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5821 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5881 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5941 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6001 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6061 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6121 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6181 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6241 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6301 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6361 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6421 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6481 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6541 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6601 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6661 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     6721 gcctctcccc gcgcgttggc cgattcatta atgcagctgt ggaatgtgtg tcagttaggg
     6781 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     6841 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     6901 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     6961 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     7021 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     7081 ctaggctttt gcaaaaagct
//
