LOCUS       sequence-459837-        4727 bp DNA     circular SYN 20-MAR-2025
DEFINITION  Single stranded AAV genome with barcodeless T7 RNA polymerase 
            promoter and SP6-driven barcode, for tracking AAV concatemers via 
            SpECTr. Also expresses mRuby2 from CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4727)
  AUTHORS   Coughlin GM, Borsos M, Barcelona BH, Appling N, Mayfield AMH, Mackey
            ED, Eser RA, Jackson CR, Chen X, Kumar SR, Gradinaru V
  TITLE     Spatial genomics of AAV vectors reveals mechanism of transcriptional
            crosstalk that enables targeted delivery of large genetic cargo
  JOURNAL   Nat Biotechnol (2025).
REFERENCE   2  (bases 1 to 4727)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 20, 2025 from SnapGene Server 7.0.3
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol (2025)."
FEATURES             Location/Qualifiers
     source          1..4727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..791
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   662..791
                     /label=AAV2 ITR
     promoter        complement(794..812)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        863..881
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     863..880
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        1027..1230
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1181..1201
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1260..1392
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1399..1408
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1405..2115
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     complement(2181..2201)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        2611..2629
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   2640..2780
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2640..2769
                     /label=AAV2 ITR
     rep_origin      2855..3310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2942..2961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3152..3173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3327..3346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3446..3468
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3506..3524)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3592..3696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3697..4557
                     /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(3915..3934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc tcgggcaaag cccgggcgtc gggcgacctt
      721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      781 taggggttcc tgccctatag tgagtcgtat tacccggcca ggaaccgtaa aaaggacttg
      841 cccgcgttgc tggcgtttaa cgatttaggt gacactatag aatacacggg tggtaaccgt
      901 attcattgga tttatgtgta tttgccagtg atggcaaatt gccaaggctg ctacggacga
      961 tgtggaagat ggcccaagag catctggaga gacaaggccg cctcgagctt gatatcgaat
     1021 tcctgcgtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     1081 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     1141 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     1201 gtacggtggg aggtctatat aagcagagct agagatttaa gaataagggc ttattacagg
     1261 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1321 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1381 ttctctccac agaccggtgc caccatggtg agcaagggag aggaactcat caaggagaac
     1441 atgcgcatga aggtagtcat ggaagggtct gtgaatgggc atcagttcaa gtgcacaggg
     1501 gaaggtgaag ggaatcccta catgggtact cagaccatga ggatcaaggt tattgaaggt
     1561 ggccctctcc cgtttgcctt cgacattctt gccactagct tcatgtatgg cagtcggacc
     1621 ttcatcaagt accccaaagg cattccagac ttcttcaaac agtcctttcc agagggcttc
     1681 acatgggaaa gggtgacccg ttacgaggat ggtggtgtgg tcacagtcat gcaggacact
     1741 agtctggagg acggatgtct ggtctatcac gtgcaagtga gaggggtgaa ctttccctcc
     1801 aatgggcctg tgatgcagaa gaaaaccaaa gggtgggagc caaacacaga gatgatgtat
     1861 cctgcagatg gaggactgag aggctatacg cacatggctc tgaaggttga cggaggaggc
     1921 cacttgtcat gctcatttgt gaccacgtac aggagcaaga aaactgtcgg caacatcaaa
     1981 atgcctggca tacatgcggt tgatcaccgc ttggaacgac tggaggagtc tgacaacgag
     2041 atgtttgtgg tacaacggga acatgcagtt gccaagtttg ctggccttgg cggaggaatg
     2101 gatgagctgt acaaatgaac gcgtcataat caacctctgg attacaaaat ttgtgaaaga
     2161 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     2221 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     2281 tggttagttc ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     2341 gctcggctgt tgggcactga caattccgtg gtgtttattt gtgaaatttg tgatgctatt
     2401 gctttatttg taaccatcta gctttatttg tgaaatttgt gatgctattg ctttatttgt
     2461 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     2521 ggttcagggg gagatgtggg aggtttttta aacaaggtac cgttaaacgc cagcaacgcg
     2581 ggcaagtcct ttttacggtt cctggccggg taatacgact cactataggc ttgggccgca
     2641 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     2701 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     2761 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2821 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     2881 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     2941 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3001 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3061 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3121 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3181 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     3241 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3301 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3361 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3421 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3481 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3541 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3601 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3661 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3721 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3781 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3841 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3901 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3961 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4021 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4081 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4141 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4201 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4261 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4321 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4381 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     4441 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4501 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4561 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4621 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4681 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
