LOCUS       sequence-309103-        6674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid with Ple32 (CLDN5 MiniPromoter) driving expression of 
            EmGFP. Contains WPRE..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6674)
  AUTHORS   Korecki AJ, Cueva-Vargas JL, Fornes O, Agostinone J, Farkas RA, 
            Hickmott JW, Lam SL, Mathelier A, Zhou M, Wasserman WW, Di Polo A, 
            Simpson EM
  TITLE     Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, 
            corneal, endothelial, Muller glial, and PAX6 cells.
  JOURNAL   Gene Ther. 2021 Feb 2. pii: 10.1038/s41434-021-00227-z. doi: 
            10.1038/s41434-021-00227-z.
  PUBMED    33531684
REFERENCE   2  (bases 1 to 6674)
  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"; journalName: "Gene Ther. 
            2021 Feb 2. pii: 10.1038/s41434-021-00227-z. doi: 
            10.1038/s41434-021-00227-z."
COMMENT     Sequence Label: pEMS2181
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     intron          1913..2045
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2028..2047
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      2066..2075
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2072..2074,2075..2077,2078..2791)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2117..2138)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2378..2397)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2725..2746
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2857..2877)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3275..3286)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3407..3528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3418..3437)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3472..3491
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3724..3853
                     /label=AAV2 ITR
     primer_bind     complement(3872..3889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3872..3888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3881..3903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4030..4485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4117..4136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4327..4348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4511..4615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4616..4684,4685..5476)
                     /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(4834..4853)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5647..6235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6136..6155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6523..6544
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6559..6564,6565..6582,6583..6589)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6597..6613
                     /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     6602..6624
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6621..6637
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6621..6637
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagatcg gaattcccta ggccggccct tgccttcaga acctccccac actaggtgag
      241 ccagacgctg gccttatctc atttaccatc tcagagccat ctgaaggggg agaagggaac
      301 cgggccccag gagggagaaa gtcatcaaac ctcccacatc tgtgaccagc ctcagtgcca
      361 tactttttct atggagggcc ctgtccaatg gaactgagca cagaccagat aaaagaactg
      421 ggcacccagt ggcctcagtc cagggcctgg agttcaaact ttactggaaa caaaggggcc
      481 gagagagact ggggaaagaa ctactagaaa ggggctggtg cccccatggg gctgtgggtt
      541 ttggagccgc gtgcccccac ctgagcctca gggggcccgg agtgtccaca ccagtggacc
      601 tttcgagaaa tggctgggcc attgtgcaga agaatgcccg gaaatcccgc gcctccctcc
      661 tccagcaagg atgggggctc ttcctcctgg ccaggaaact ccaagttggc ttccggaggg
      721 tggcctgggg gctggggtgc cagggacacc atcgccactg gtgggagggc agggcacagc
      781 ccctccgtgt ccctttgtct ctcctgtctg aaggccagag caggctgcta ggcctggggc
      841 caccactgcc cctgggtgct acacccagtg tgctgggtca ctgggaactt cctgaagtgg
      901 tgtcacctga actgggcccc caaggatggg gtgcgggcag taccgcagga agaggagcag
      961 cccctgtgaa gattgagagg tctgggaagc ccctgcggct tgggagagtg ggggtcgcca
     1021 ggcaggggga aagcccctgt gccaccgctt tttgccagag actcaggctc cagagaggca
     1081 gtgagtggca tggggggtga ggctggggcc ctgggcctga cctccacacg cctgcctggc
     1141 ctctctgttt gccatgggat gagagagaca gtgctgggac tcagagcggg gctggagagt
     1201 gagagtgcga gaaagggcct gggtggggct tggaccccgg ggcgggcttt ctggagagcc
     1261 cccctacgag ggcctctacg gcggtgacgg ggtggggggc ttctgcaaac cttggtcagg
     1321 gaagtggagc tggctcgagt ggaagagacc acccggctca gtcggggatg tgggagtgga
     1381 ctgggtggtg cagactgggg gtcgagcgcc ttctgaagtg acggggccgg gacgcgcagg
     1441 gaggcggccc aagaagcgcg ccctaggcca gcccagaatg cgctcggccg cgactaggac
     1501 aacggcgggt ggggctgggg gcggctgccg ggcggggagc ggtcccgcgc cctcagctac
     1561 ccctcaagag ccgttgtttc cctaacttca gctgccagag gctctgtgat tggctgcggc
     1621 acgatgaccc gcgcacggat tggctgcttc gggccggggg gccgggcccg ggggacagaa
     1681 tccgcccccg aaccttcaaa gagggtaccc cccggcagga gctggcagac ccaggaggtg
     1741 cgacagaccc gcggggcaaa cggactgggg ccaagagccg ggagcgcggg cgcaaaggca
     1801 ccagggcccg cccagggcgc cgcgcagcac ggccttgggg gttctgcggg ccttcgggtg
     1861 cgcgtctcgc ctctagccgg cgcgccgaag ttggtcgtga ggcactgggc aggtaagtat
     1921 caaggttaca agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag
     1981 actcttgcgt ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc
     2041 cacaggtgtc cactcccagg cggccgccac catggtgagc aagggcgagg agctgttcac
     2101 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2161 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2221 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca
     2281 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2341 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2401 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2461 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2521 ggtctatatc accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca
     2581 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2641 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2701 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2761 cactctcggc atggacgagc tgtacaagta agcggccgcg atcgctcaac ctctggatta
     2821 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2881 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2941 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     3001 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3061 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3121 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3181 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     3241 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3301 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3361 gagtcggatc tccctttggg ccgcctcccc gcgatcgcag acatgataag atacattgat
     3421 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     3481 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     3541 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa
     3601 aacctctaca aatgtggtag tcgaaattcc cgataaggat cttcgtcgac gaattcccga
     3661 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact
     3721 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     3781 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     3841 agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact
     3901 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3961 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4021 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4081 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4141 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4201 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4261 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4321 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4381 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4441 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     4501 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4561 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4621 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4681 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4741 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4801 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4861 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4921 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4981 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5041 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5101 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5161 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5221 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5281 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5341 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5401 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5461 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5521 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5581 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5641 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5701 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5761 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5821 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5881 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5941 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6001 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6061 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6121 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6181 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6241 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6301 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     6361 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     6421 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     6481 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     6541 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     6601 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca gatttaatta
     6661 aggccttaat tagg
//
