LOCUS       sequence-481883-        6189 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  AAV construct for constitutive expression of EGFP. Contains a 
            molecular barcode for identification of AAV genome using single-cell
            sequencing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6189)
  AUTHORS   Belfort BDW, Jia JD, Garza AR, Insalaco AM, McGinnis JP, Pekarek BT,
            Ortiz-Guzman J, Tepe B, Chen H, Liu Z, Arenkiel BR
  TITLE     Comparative analysis of AAV serotypes for transduction of olfactory 
            sensory neurons.
  JOURNAL   Front Neurosci. 2025 Feb 13;19:1531122. doi: 
            10.3389/fnins.2025.1531122. eCollection 2025.
  PUBMED    40018362
REFERENCE   2  (bases 1 to 6189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front 
            Neurosci."; date: "2025-02-13"; volume: "19:1531122. doi"; pages: " 
            10.3389/fnins.2025.1531122. eCollection 2025"
COMMENT     Sequence Label: rAAV-Ef1-alpha-EGFP-WPRE-ID-PolyA
FEATURES             Location/Qualifiers
     source          1..6189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      187..642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(274..293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     484..505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(659..678)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     778..800
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(838..856)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        924..1028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1029..1097,1098..1889)
                     /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(1247..1266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2060..2648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2549..2568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2710..2839
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2940..4118
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3171..4109
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4066..4086
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4182..4238
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(4242..4244,4245..4247,4248..4961)
                     /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(4242..4244,4245..4247,4248..4958)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4287..4308)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4548..4567)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4895..4916
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5000..5588
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5053..5073)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5471..5482)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5645..6121
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5844..5863)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   join(6161..6189,1..112)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(6161..6189,1..101)
                     /label=AAV2 ITR
ORIGIN
        1 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
       61 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
      121 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
      181 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      241 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
      301 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      361 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
      421 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      481 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
      541 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
      601 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
      661 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
      721 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      781 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
      841 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
      901 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      961 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1021 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1081 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1141 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1201 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     1261 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     1321 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     1381 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     1441 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     1501 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     1561 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     1621 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     1681 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     1741 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     1801 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     1861 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1921 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1981 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2041 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2101 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2161 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     2221 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2281 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2341 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2401 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2461 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2521 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2581 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2641 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2701 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
     2761 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     2821 tccatcacta ggggttcctg cggccgcacg cgtaagcttt gcaaagatgg ataaagtttt
     2881 aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga gtgggaattg
     2941 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     3001 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     3061 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     3121 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     3181 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     3241 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     3301 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     3361 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     3421 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     3481 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     3541 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     3601 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     3661 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     3721 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     3781 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     3841 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     3901 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     3961 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     4021 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     4081 tggttcaaag tttttttctt ccatttcagg tgtcgtgagg taccggatca gcttggatcc
     4141 actagtttaa ttaatctaga gtcgacgcta gcggtagtgg agccaccaat ttctccctcc
     4201 tgaaacaggc aggagacgtc gaggagaatc ctggaccatc catggtgagc aagggcgagg
     4261 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4321 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4381 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     4441 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     4501 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     4561 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4621 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4681 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     4741 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4801 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     4861 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4921 ccgccgggat cactctcggc atggacgagc tgtacaagta gcctaggggc gcgccgaatt
     4981 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     5041 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     5101 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     5161 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     5221 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     5281 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     5341 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     5401 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     5461 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     5521 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     5581 ctccccgcat cgataccgag ccgacgcgtt ttaccttgtg gattttgctg ctcgagagat
     5641 ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     5701 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     5761 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     5821 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     5881 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     5941 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     6001 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     6061 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     6121 tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     6181 ttggccact
//
