LOCUS       sequence-378756-        6958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Nanoluc-P2A-tdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6958)
  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 6958)
  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-tdT
FEATURES             Location/Qualifiers
     source          1..6958
                     /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             2031..3461
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGIGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     misc_feature    3478..4066
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3531..3551)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3949..3960)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4087..4168
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4124..4143)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4178..4197
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4221..4361
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4221..4350
                     /label=AAV2 ITR
     rep_origin      4436..4891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4523..4542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4733..4754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4908..4927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5027..5049
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5087..5105)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5173..5277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5278..5346,5347..6138)
                     /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(5496..5515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6309..6897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6798..6817
                     /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 tcccgggcca atggtgagca
     2041 agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag ggctccatga
     2101 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     2161 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     2221 cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc cccgattaca
     2281 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     2341 gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc tacaaggtga
     2401 agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag accatgggct
     2461 gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccacc
     2521 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc atctacatgg
     2581 ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg gacatcacct
     2641 cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc cgccaccacc
     2701 tgttcctggg gcatggcatc ggcagcaccg gcagcggcag ctccggcacc gcctcctccg
     2761 aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
     2821 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     2881 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
     2941 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
     3001 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     3061 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
     3121 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
     3181 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
     3241 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct
     3301 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
     3361 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
     3421 accacctgtt cctgtacggc atggacgagc tgtacaagta ataagaattc tatcgataat
     3481 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3541 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3601 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3661 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3721 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3781 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3841 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3901 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3961 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     4021 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataagtaagt
     4081 gactagaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4141 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatctgc
     4201 acgcgtggcg cgccggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4261 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4321 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4381 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4441 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4501 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4561 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4621 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4681 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4741 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     4801 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4861 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4921 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4981 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5041 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5101 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5161 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5221 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5281 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5341 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5401 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5461 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5521 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5581 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5641 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5701 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5761 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5821 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5881 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5941 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6001 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6061 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6121 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6181 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6241 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6301 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6361 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6421 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6481 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6541 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6601 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6661 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6721 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6781 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6841 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6901 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
