LOCUS       sequence-481895-        5730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tested on HeLa for bioluminescence, and used for production of AAV 
            G12-Akaluc-P2A-Proinsulin for mice..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5730)
  AUTHORS   Kasatkina LA, Ma C, Sheng H, Lowerison M, Menozzi L, Baloban M, Tang
            Y, Xu Y, Humayun L, Vu T, Song P, Yao J, Verkhusha VV
  TITLE     Deep-tissue high-sensitivity multimodal imaging and optogenetic 
            manipulation enabled by biliverdin reductase knockout.
  JOURNAL   Nat Commun. 2025 Jul 14;16(1):6469. doi: 10.1038/s41467-025-61532-4.
  PUBMED    40659617
REFERENCE   2  (bases 1 to 5730)
  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 
            Commun."; date: "2025-07-14"; volume: "16(1):6469. doi"; pages: " 
            10.1038/s41467-025-61532-4"
COMMENT     Sequence Label: pAAV-G12-AkaLuc-P2A-Proinsulin
FEATURES             Location/Qualifiers
     source          1..5730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        185..203
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     185..202
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      511..520
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2182..2238
                     /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"
     primer_bind     complement(2662..2682)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     2864..2883
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2993..3133
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2993..3122
                     /label=AAV2 ITR
     rep_origin      3208..3663
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3295..3314)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3505..3526
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3680..3699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3799..3821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3859..3877)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3945..4049
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4050..4118,4119..4910)
                     /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(4268..4287)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5081..5669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5570..5589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgaag atgctgcaca cttgtgggct agccttatgt atcatacaca
      181 tacgatttag gtgacactat agaactcgag cagctgaagc ttcggagtac tgtcctccga
      241 gcggagtact gtcctccgag cggagtactg tcctccgagt cgactctagc ggagtactgt
      301 cctccgagcg gagtactgtc ctccgagcgg agtactgtcc tccgagtcga ctctagcgga
      361 gtactgtcct ccgagcggag tactgtcctc cgagcggagt actgtcctcc gagtcgactc
      421 tagcggagta ctgtcctccg agcggagtac tgtcctccgg ctagtagcgg agactctaga
      481 gggtatataa tggatccccg ggtaccggtc gccaccatgg aagatgccaa aaacattaag
      541 aagggcccag cgccgttcta cccactcgaa gacgggaccg ccggcgagca gctgcacaaa
      601 gccatgaagc gctacgccct ggtgcccggc gccatcgcct ttaccgacgc acatattcag
      661 gtggacgtta cctacgccga gtacttcgag atgagcgttc ggctggcaga agctatgagg
      721 cgctatgggc tgaatacaaa ccatcggatc gtggtgtgca gcgagaatag ctcgcagttc
      781 ttcatgcccg tgttgggtgc cctgttcatc ggtgtggctg tggccccagc taacgacatc
      841 tacaacgagc gcgagctgct gaacagcatg ggcatcagcc agcccaccgt cgtattcgtg
      901 agcaagaaag ggctgcgaaa ggtcctcaac gtgcaaaaga agctaccgat catacgaaag
      961 atcatcatca tggatagcaa gaccgactac cagggcttcc aaagcatgta caccttcgtg
     1021 acttcccatt tgccacccag cttcaacgag tacgacttcg tgcccgagag cttcgaccgg
     1081 gacaaaacca tcgccctgat catgaacagt agtggtagta caggattacc caagggcgta
     1141 gccctaccgc accgcaccgc ttgtgtccga ttcagtcatg cccgcgaccc catcttcggc
     1201 taccagaaca tccccgacac cgctatcctc agcgtggtgc catttcacca cggcttcggc
     1261 atgttcacca cgctgggcta cttgatctgc ggctttcggg tcgtgctcat gtaccgcttc
     1321 gaggaggagc tattcttgcg cagcttgcaa gactataaga ttcaatctgc cctgctggtg
     1381 cccacactat ttagctgcct cgctaagagc actctcatcg acaagtacga cctaagcagc
     1441 ttgcgcgaga tcgccagcgg cggggcgccg ctcagcaagg aggtaggtga ggccgtggcc
     1501 aaacgcttcc gcctaccagg catccgccag ggctatggcc tgacagaaac aaccaacgcc
     1561 gtcatgatca cccccgaggg ggaccgtaag cctggctcag taggcaaggt ggtgcccttc
     1621 ttcgaggcta aggtggtaga cttggtcacc ggtaagacac tgggtgtgaa ccagcgcggt
     1681 gagctgtgcg tccgtggccc catgatcatg agcggctacg ttaacaaccc cgaggctacg
     1741 aacgctctca tcgacaagga cggctggctg cacagcggcg acatcgccta ctgggacgag
     1801 gacgagcact tcttcatcgt ggaccggctg aagagcctga tcaaatacaa gggctaccag
     1861 gtagccccag ccgaactgga gggcatcctg ctgcaacacc cctacatctt cgacgccgga
     1921 gtcgccggcc tgcccgacga cgatgccggc gagctgcccg ccgcagtcgt cgtgttggaa
     1981 cacggtaaaa ccatgaccga gaaagagatc gtggactatg tggccagcca ggttacaacc
     2041 gccaagaagc tgcgcggtgg tgttgtgttt gtggatgaag tccctagagg atcgaccggc
     2101 aagttagacg cccgcaagat ccgcgagatt ctcactaagg ccaagaagga cggcaagatc
     2161 gccgtgagat ctggctcagg ggccaccaac ttcagcctgc taaagcaggc tggggatgtg
     2221 gaagaaaatc caggaccttc aggaatggcc ctctggatga ggtttctccc gttgctggcc
     2281 ttgctcgttc tgtgggagcc caagccagcc caggcatttg tcaaacaaca tctttgtggc
     2341 cctcacctgg tggaggcgtt atatttggtt tgtggagagc gaggcttctt ctatactcca
     2401 aagtctcgca ggaaaagaga agacccccag gtccctcaac ttgagctggg cggtggtcct
     2461 gaggcaggag acctgcagac gctagcactg gaggtggccc ggcagaagag aggcattgtg
     2521 gaccagtgct gcacatccat ctgtagtctc taccagctgg aaaactactg caattaagcg
     2581 gccgcgactc tagaattccg ctcgagataa tcaacctctg gattacaaaa tttgtgaaag
     2641 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     2701 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     2761 ctggttagtt cttgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     2821 ggctcggctg ttgggcactg acaattccgt ggtgtttatt tgtgaaattt gtgatgctat
     2881 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2941 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcggcc gcaggaaccc
     3001 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3061 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3121 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3181 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     3241 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     3301 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3361 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3421 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3481 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     3541 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     3601 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3661 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3721 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3781 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3841 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     3901 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3961 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4021 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4081 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4141 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4201 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4261 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4321 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4381 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4441 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4501 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4561 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     4621 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4681 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4741 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4801 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4861 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4921 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4981 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5041 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5101 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5161 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5221 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5281 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5341 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5401 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5461 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5521 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5581 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5641 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5701 tggccttttg ctggcctttt gctcacatgt
//
