LOCUS       sequence-378645-        6282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Nanoluc-P2A-copGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6282)
  AUTHORS   Kulkarni AA, Seal AG, Sonnet C, Oka K
  TITLE     Streamlined Adeno-Associated Virus Production Using Suspension 
            HEK293T Cells.
  JOURNAL   Bio Protoc. 2024 Feb 5;14(3):e4931. doi: 10.21769/BioProtoc.4931. 
            eCollection 2024 Feb 5.
  PUBMED    38379831
REFERENCE   2  (bases 1 to 6282)
  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: "Bio 
            Protoc."; date: "2024-02-5"; volume: "14(3):e4931. doi"; pages: " 
            10.21769/BioProtoc.4931. eCollection 2024 Feb 5"
COMMENT     Sequence Label: AV0-EF1-N-cG
FEATURES             Location/Qualifiers
     source          1..6282
                     /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        243..1421
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          474..1412
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1369..1389
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1437..1949
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1965..2021
                     /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             2046..2708
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
     misc_feature    2802..3390
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2855..2875)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3273..3284)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3411..3492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3448..3467)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3502..3521
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3545..3685
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3545..3674
                     /label=AAV2 ITR
     rep_origin      3760..4215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3847..3866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4057..4078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4232..4251)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4351..4373
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4411..4429)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4497..4601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4602..4670,4671..5462)
                     /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(4820..4839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5633..6221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6122..6141
                     /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 gcggccggcg cgccctgcag tttcccaagc tttgcaaaga tggataaagt
      181 tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa
      241 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
      301 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
      361 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
      421 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
      481 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
      541 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
      601 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
      661 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
      721 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
      781 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
      841 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
      901 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
      961 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     1021 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     1081 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     1141 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     1201 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     1261 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     1321 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     1381 cagtggttca aagttttttt cttccatttc aggtgtcgtg aggaattgga tccaccatgg
     1441 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
     1501 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
     1561 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
     1621 tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
     1681 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
     1741 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
     1801 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
     1861 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
     1921 gctggcggct gtgcgaacgc attctggcga agcttggaag cggagctact aacttcagcc
     1981 tgctgaagca ggctggagac gtggaggaga accctggacc tatggagagc gacgagagcg
     2041 gcctgcccgc catggagatc gagtgccgca tcaccggcac cctgaacggc gtggagttcg
     2101 agctggtggg cggcggagag ggcaccccca agcagggccg catgaccaac aagatgaaga
     2161 gcaccaaagg cgccctgacc ttcagcccct acctgctgag ccacgtgatg ggctacggct
     2221 tctaccactt cggcacctac cccagcggct acgagaaccc cttcctgcac gccatcaaca
     2281 acggcggcta caccaacacc cgcatcgaga agtacgagga cggcggcgtg ctgcacgtga
     2341 gcttcagcta ccgctacgag gccggccgcg tgatcggcga cttcaaggtg gtgggcaccg
     2401 gcttccccga ggacagcgtg atcttcaccg acaagatcat ccgcagcaac gccaccgtgg
     2461 agcacctgca ccccatgggc gataacgtgc tggtgggcag cttcgcccgc accttcagcc
     2521 tgcgcgacgg cggctactac agcttcgtgg tggacagcca catgcacttc aagagcgcca
     2581 tccaccccag catcctgcag aacgggggcc ccatgttcgc cttccgccgc gtggaggagc
     2641 tgcacagcaa caccgagctg ggcatcgtgg agtaccagca cgccttcaag acccccattg
     2701 ccttcgccag atcccgcgct cagtcgtcca attctgccgt ggacggcacc gccggacccg
     2761 gctccaccgg atctcgctaa ggcgcgccga attctatcga taatcaacct ctggattaca
     2821 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     2881 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     2941 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3001 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3061 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3121 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3181 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     3241 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3301 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3361 gtcggatctc cctttgggcc gcctccccgc atcgataagt aagtgactag aacttgttta
     3421 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3481 ttttttcact gcattctagt tgtggtttgt ccaaactcat ctgcacgcgt ggcgcgccgg
     3541 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     3601 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     3661 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     3721 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     3781 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     3841 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3901 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3961 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4021 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4081 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     4141 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4201 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4261 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4321 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4381 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4441 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4501 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4561 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4621 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4681 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4741 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4801 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4861 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4921 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4981 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5041 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5101 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5161 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5221 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5281 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5341 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5401 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5461 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5521 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5761 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5821 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5881 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5941 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6001 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6061 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6121 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6181 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6241 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
