LOCUS       sequence-478216-        8426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AAV2 transgene (EGFP) and MbPylRS/tRNA for ncAA 
            incorporation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8426)
  AUTHORS   Pham Q, Glicksman J, Shahraeini S, Han B, Jewel D, Loynd C, Singha 
            Roy SJ, Chatterjee A
  TITLE     A facile chemical strategy to synthesize precise AAV-protein 
            conjugates for targeted gene delivery
  JOURNAL   bioRxiv 2024.07.20.604406
REFERENCE   2  (bases 1 to 8426)
  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: "bioRxiv 
            2024.07.20.604406"
COMMENT     Sequence Label: pIDTsmart-MbPylRS-4xPyOtR-ITR-GFP
FEATURES             Location/Qualifiers
     source          1..8426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   210..350
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   221..350
                     /label=AAV2 ITR
     polyA_signal    390..866
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     648..667
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     CDS             complement(join(925..1635,1636..1638,1639..1641))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(925..1635,1636..1638,1639..1641))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(967..988)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1316..1335
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1575..1596
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          1722..2100
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     primer_bind     complement(2228..2252)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2249..2452)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        2453..2756
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   2832..2961
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3002..3250
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3349..3597
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3696..3944
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        4043..4291
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     enhancer        4429..4732
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4733..4936
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4981..5000
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4981..4999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5019..5028
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6349..6366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6355..6579
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6727..6743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6727..6743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      6791..6822
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        6823..6925
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             6926..7741
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6999..7018)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      7765..7792
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(7804..8391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7884..7903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtgtaaa acgacggcca gtttatctag tcagcttgat tctagctgat cgtggaccgg
       61 aaggtgagcc agtgagttga ttgcagtcca gttacgctgg agtctgaggc tcgtcctgaa
      121 tgatatgcga ccgccggagg gttgcgtttg agacgggcga cagatccagt cgcgctgctc
      181 tcgtcgatcc gctagggcgg ccgcaaatac ctgcaggcag ctgcgcgctc gctcgctcac
      241 tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag
      301 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccgctc
      361 ggtccgcacg tggttaccta caaaatcaga aggacaggga agggagcagt ggttcacgcc
      421 tgtaatccca gcaatttggg aggccaaggt gggtagatca cctgagatta ggagttggag
      481 accagcctgg ccaatatggt gaaaccccgt ctctaccaaa aaaacaaaaa ttagctgagc
      541 ctggtcatgc atgcctggaa tcccaacaac tcgggaggct gaggcaggag aatcgcttga
      601 acccaggagg cggagattgc agtgagccaa gattgtgcca ctgcactcca gcttggttcc
      661 caatagaccc cgcaggccct acaggttgtc ttcccaactt gccccttgct ccataccacc
      721 cccctccacc ccataatatt atagaaggac acctagtcag acaaaatgat gcaacttaat
      781 tttattagga caaggctggt gggcactgga gtggcaactt ccagggccag gagaggcact
      841 ggggaggggt cacagggatg ccacccgtag atctctcgag cagcgctgct cgaggcaagc
      901 ttacttgagc tcgagatctg agtacttgta cagctcgtcc atgccgagag tgatcccggc
      961 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     1021 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     1081 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     1141 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     1201 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1261 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1321 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     1381 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     1441 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     1501 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     1561 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     1621 ctcctcgccc ttgctcacca ttcagaattc aatcgatgtt cgaatcccaa ttctttgcca
     1681 aagtgatggg ccagcacaca gaccagcacg ttgcccagga gctgtgggag gaagataaga
     1741 ggtatgaaca tgattagcaa aagggcctag cttggactca gaataatcca gccttatccc
     1801 aaccataaaa taaaagcaga atggtagctg gattgtagct gctattagca atatgaaacc
     1861 tcttacatca gttacaattt atatgcagaa atatttatat gcagaaatat tgctattgcc
     1921 ttaacccaga aattatcact gttattcttt agaatggtgc aaagaggcat gatacattgt
     1981 atcattattg ccctgaaaga aagagattag ggaaagtatt agaaataaga taaacaaaaa
     2041 agtatattaa aagaagaaag cattttttgt gggcctatag actctatagg cggtacttac
     2101 gtcactcttg gcacggggaa tccgcgttcc aatgcaccgt tcccggccgg gattcgaatc
     2161 cgcggaggct ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc
     2221 tccaggcgat ctgacggttc actaaacgag ctctgcttat atagacctcc caccgtacac
     2281 gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt
     2341 gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc
     2401 gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa
     2461 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
     2521 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     2581 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     2641 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     2701 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     2761 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagacg
     2821 cgtgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     2881 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc
     2941 gagcgcgcag ctgcctgcag gctcgatccg ctcgcacccc taggggtacc tcgggcagga
     3001 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     3061 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     3121 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     3181 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     3241 acgaaacacc gggcggctga tcatgtagat cgaacggact ctaaatccgt tcagccgggt
     3301 tagattcccg ggctgcccgt tttttgctag gggtacctcg ggcaggaaga gggcctattt
     3361 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
     3421 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     3481 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     3541 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg
     3601 cggctgatca tgtagatcga acggactcta aatccgttca gccgggttag attcccgggc
     3661 tgcccgtttt ttgctagggg tacctcgggc aggaagaggg cctatttccc atgattcctt
     3721 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     3781 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     3841 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     3901 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggcgg ctgatcatgt
     3961 agatcgaacg gactctaaat ccgttcagcc gggttagatt cccgggctgc ccgttttttg
     4021 ctaggggtac ctcgggcagg aagagggcct atttcccatg attccttcat atttgcatat
     4081 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
     4141 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     4201 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     4261 tttatatatc ttgtggaaag gacgaaacac cgggcggctg atcatgtaga tcgaacggac
     4321 tctaaatccg ttcagccggg ttagattccc gggctgcccg ttttttgcta gttattaata
     4381 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     4441 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     4501 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     4561 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     4621 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     4681 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     4741 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     4801 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     4861 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     4921 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     4981 taatacgact cactataggg agacccaagc tggctagcgc caccatggat aaaaaaccat
     5041 tagatgtttt aatatctgcg accgggctct ggatgtccag gactggcacg ctccacaaaa
     5101 tcaagcacca tgaggtctca agaagtaaaa tatacattga aatggcgtgt ggagaccatc
     5161 ttgttgtgaa taattccagg agttgtagaa cagccagagc attcagacat cataagtaca
     5221 gaaaaacctg caaacgatgt agggtttcgg acgaggatat caataatttt ctcacaagat
     5281 caaccgaaag caaaaacagt gtgaaagtta gggtagtttc tgctccaaag gtcaaaaaag
     5341 ctatgccgaa atcagtttca agggctccga agcctctgga aaattctgtt tctgcaaagg
     5401 catcaacgaa cacatccaga tctgtacctt cgcctgcaaa atcaactcca aattcgtctg
     5461 ttcccgcatc ggctcctgct ccttcactta caagaagcca gcttgatagg gttgaggctc
     5521 tcttaagtcc agaggataaa atttctctaa atatggcaaa gcctttcagg gaacttgagc
     5581 ctgaacttgt gacaagaaga aaaaacgatt ttcagcggct ctataccaat gatagagaag
     5641 actacctcgg taaactcgaa cgtgatatta cgaaattttt cgtagaccgg ggttttctgg
     5701 agataaagtc tcctatcctt attccggcgg aatacgtgga gagaatgggt attaataatg
     5761 atactgaact ttcaaaacag atcttccggg tggataaaaa tctctgcttg aggccaatgc
     5821 ttgccccgac tctttacaac tatctgcgaa aactcgatag gattttacca ggcccaataa
     5881 aaattttcga agtcggacct tgttaccgga aagagtctga cggcaaagag cacctggaag
     5941 aatttactat ggtgaacttc tgtcagatgg gttcgggatg tactcgggaa aatcttgaag
     6001 ctctcatcaa agagtttctg gactatctgg aaatcgactt cgaaatcgta ggagattcct
     6061 gtatggtctt tggggatact cttgatataa tgcacgggga cctggagctt tcttcggcag
     6121 tcgtcgggcc agtttctctt gatagagaat ggggtattga caaaccatgg ataggtgcag
     6181 gttttggtct tgaacgcttg ctcaaggtta tgcacggctt taaaaacatt aagagggcat
     6241 caaggtccga atcttactat aatgggattt caaccaatct gtaagaattc aacgcgttaa
     6301 gtcgacttta actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     6361 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     6421 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     6481 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     6541 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaaccc
     6601 taggggtgcg agcggatcga gcagtgtcga tcactactgg accgcgagct gtgctgcgac
     6661 ccgtgatctt acggcattat acgtatgatc ggtccacgat cagctagatt atctagtcag
     6721 cttgatgtca tagctgtttc ctgaggctca atactgacca tttaaatcat acctgacctc
     6781 catagcagaa agtcaaaagc ctccgaccgg aggcttttga cttgatcggc acgtaagagg
     6841 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttatcgag
     6901 attttcagga gctaaggaag ctaaaatgag ccatattcaa cgggaaacgt cttgcttgaa
     6961 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     7021 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
     7081 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcaggct
     7141 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     7201 tgatgcatgg ttactcacca ctgcgatccc agggaaaaca gcattccagg tattagaaga
     7261 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     7321 ttcgattcct gtttgtaatt gtccttttaa cggcgatcgc gtatttcgtc tcgctcaggc
     7381 gcaatcacga atgaataacg gtttggttgg tgcgagtgat tttgatgacg agcgtaatgg
     7441 ctggcctgtt gaacaagtct ggaaagaaat gcataaactc ttgccattct caccggattc
     7501 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     7561 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     7621 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     7681 tattgataat cctgatatga ataaattgca gtttcacttg atgctcgatg agtttttcta
     7741 atgaggacct aaatgtaatc acctggctca ccttcgggtg ggcctttctg cgttgctggc
     7801 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgatgct caagtcagag
     7861 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7921 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7981 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     8041 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     8101 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     8161 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     8221 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     8281 tacctcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     8341 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     8401 ttgattttct accgaagaaa ggccca
//
