LOCUS       sequence-387121-        5991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of BDFP1.6-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5991)
  AUTHORS   Zhang H, Papadaki S, Sun X, Wang X, Drobizhev M, Yao L, Rehbock M, 
            Koster RW, Wu L, Namikawa K, Piatkevich KD
  TITLE     Quantitative assessment of near-infrared fluorescent proteins.
  JOURNAL   Nat Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z.
  PUBMED    37666982
REFERENCE   2  (bases 1 to 5991)
  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 
            Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z."
COMMENT     Sequence Label: pAAV-CAG-BDFP1.6-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..558
                     /label=chicken beta-actin promoter
     primer_bind     837..859
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     921..940
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      979..988
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1465..1521
                     /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             join(1534..1536,1537..2250)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1534..1536,1537..2247)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1576..1597)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1837..1856)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2184..2205
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2275..2863
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2328..2348)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2746..2757)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2906..3027
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2917..2936)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2971..2990
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3189..3318
                     /label=AAV2 ITR
     rep_origin      complement(3555..4143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3635..3654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4314..5105,5106..5174))
                     /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     4937..4956
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5175..5279)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5305..5760)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5442..5463)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5654..5673
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5832..5961
                     /label=AAV2 ITR
ORIGIN
        1 tatctacgta gccatgctct aggaagagta ccattgacgt caataatgac gtatgttccc
       61 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      121 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      181 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      241 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      301 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      361 taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg
      421 gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      481 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      541 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      601 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      661 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      721 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      781 ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
      841 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
      901 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
      961 agaattggat ccggtaccgc caccatggcc aatcgcgagg ttgttgagac cctgaaggag
     1021 ctcctggctg acggggagaa gagagtgcag gtggctggag tgattgggac taacgctgcc
     1081 gaagttgtca agaccgccgt gtcacttctg tttcaggagt acccagagtt ggtgtcccct
     1141 ggcgggtgtg cctatacaac aaggcgctac aacatgtgtg tgagggacat gaactacttc
     1201 ctcaggatgt gtagctacgc catcgtggcc gggggcgcca gcgtcttgga tggaaggatg
     1261 ctggccgggt tccgggatac attcaactcc ctcggcatcc ctctgtgccc cgctgcccgg
     1321 ggcatccaac tgatgaagaa gatcgtcaaa gagaaactgg cgacagccgg catgactaac
     1381 attgcatttg tcgatgagcc ctttgactac atcgcccgtg tgattagcga aacggaaatt
     1441 cgaccggttc acgtgggaag tggggctaca aacttctcac tgctgaaaca ggccggggat
     1501 gtggaggaaa acccaggacc acacgtgact agtgtgagca agggcgagga gctgttcacc
     1561 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1621 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1681 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1741 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1801 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1861 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1921 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1981 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2041 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2101 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2161 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2221 actctcggca tggacgagct gtacaagtaa gaattcgata tcaagcttat cgataatcaa
     2281 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     2341 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     2401 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     2461 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     2521 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     2581 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     2641 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     2701 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     2761 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     2821 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc
     2881 gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     2941 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3001 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3061 gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     3121 atcgataagg atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat
     3181 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3241 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     3301 gagcgagcga gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3361 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3421 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3481 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3541 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3601 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3661 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3721 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3781 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3841 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3901 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3961 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4021 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4081 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4141 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4201 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4261 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4321 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4381 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4441 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4501 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4561 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4621 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4681 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4741 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4801 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4861 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4921 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4981 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5041 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5101 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5161 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5221 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5281 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     5341 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     5401 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     5461 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     5521 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     5581 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     5641 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     5701 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     5761 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     5821 tattacgcca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
     5881 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     5941 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac t
//
