LOCUS       sequence-375404-        6316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of a Cre reporter with EYFP output.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6316)
  AUTHORS   Kaseniit KE, Katz N, Kolber NS, Call CC, Wengier DL, Cody WB, 
            Sattely ES, Gao XJ
  TITLE     Modular, programmable RNA sensing using ADAR editing in living 
            cells.
  JOURNAL   Nat Biotechnol. 2022 Oct 5. pii: 10.1038/s41587-022-01493-x. doi: 
            10.1038/s41587-022-01493-x.
  PUBMED    36198772
REFERENCE   2  (bases 1 to 6316)
  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: "Nat 
            Biotechnol. 2022 Oct 5. pii: 10.1038/s41587-022-01493-x. doi: 
            10.1038/s41587-022-01493-x."
COMMENT     Sequence Label: EK0312 pAAV-EF1a-DIO-EYFP-WPRE-HGHpA
FEATURES             Location/Qualifiers
     source          1..6316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        232..1410
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          463..1401
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1358..1378
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1441..1474)
                     /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(1525..1558)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1570..2283,2284..2286,2287..2289))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1615..1636)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1964..1983
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2223..2244
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2286..2295
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2302..2335
                     /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    2386..2419
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2444..3032
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2497..2517)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2915..2926)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3064..3540
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3263..3282)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3580..3720
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3580..3709
                     /label=AAV2 ITR
     rep_origin      3795..4250
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3882..3901)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4092..4113
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4267..4286)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4386..4408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4446..4464)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4532..4636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4637..4705,4706..5497)
                     /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(4855..4874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5668..6256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6157..6176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
      181 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      241 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      301 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      361 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      421 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      481 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      541 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      601 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      661 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      721 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      781 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      841 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
      901 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
      961 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1021 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1081 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1141 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1201 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1261 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1321 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1381 agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt cgactccgga
     1441 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
     1501 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
     1561 cgcgccctat tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     1621 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactggta
     1681 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     1741 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac
     1801 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
     1861 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
     1921 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
     1981 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
     2041 gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac tgcaggccgt agccgaaggt
     2101 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
     2161 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
     2221 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     2281 gctcaccatg gtggcgctag cataacttcg tataaagtat cctatacgaa gttatttgcc
     2341 ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat
     2401 gtatgctata cgaagttatg aattcgatat caagcttatc gataatcaac ctctggatta
     2461 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2521 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2581 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2641 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2701 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     2761 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2821 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     2881 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     2941 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3001 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     3061 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     3121 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     3181 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     3241 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     3301 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     3361 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     3421 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     3481 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     3541 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     3601 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     3661 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     3721 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     3781 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3841 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     3901 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3961 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4021 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4081 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     4141 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     4201 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4261 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4321 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4381 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4441 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4501 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4561 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4621 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4681 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4741 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4801 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4861 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4921 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4981 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5041 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5101 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5161 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5221 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5281 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5341 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5401 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5461 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5521 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5581 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5641 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5701 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5761 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5821 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5881 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5941 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6001 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6061 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6121 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6181 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6241 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6301 gccttttgct cacatg
//
