LOCUS       sequence-387120-        6334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of iRFP682-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6334)
  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 6334)
  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-iRFP682-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6334
                     /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             985..1932
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 682 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP682
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGVVIAEDRCAEVESKLGLHYPASAVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPCHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     CDS             1957..2013
                     /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(2026..2028,2029..2742)
                     /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(2026..2028,2029..2739)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2068..2089)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2329..2348)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2676..2697
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2767..3355
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2820..2840)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3238..3249)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3398..3519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3409..3428)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3463..3482
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3898..4486)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3978..3997)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4657..5448,5449..5517))
                     /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     5280..5299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5518..5622)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5648..6103)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5785..5806)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5997..6016
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6175..6304
                     /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 caccatggcg gaaggatccg tcgccaggca gcctgacctc
     1021 ttgacctgcg acgatgagcc gatccatatc cccggtgcca tccaaccgca tggactgctg
     1081 ctcgccctcg ccgccgacat gacgatcgtt gccggcagcg acaaccttcc cgaactcacc
     1141 ggactggcga tcggcgccct gatcggccgc tctgcggccg atgtcttcga ctcggagacg
     1201 cacaaccgtc tgacgatcgc cttggccgag cccggggcgg ccgtcggagc accgatcact
     1261 gtcggcttca cgatgcgaaa ggacgcaggc ttcatcggct cctggcatcg ccatgatcag
     1321 ctcatcttcc tcgagctcga gcctccccag cgggacgtcg ccgagccgca ggcgttcttc
     1381 cgccgcacca acagcgccat ccgccgcctg caggccgccg aaaccttgga aagcgcctgc
     1441 gccgccgcgg cgcaagaggt gcggaagatt accggcttcg atcgggtgat gatctatcgc
     1501 ttcgcctccg acttcagcgg ggtggtgatc gcagaggatc gatgcgccga ggtcgagtca
     1561 aaactaggcc tgcactatcc tgcctcagcg gtgccggcgc aggcccgtcg gctctatacc
     1621 atcaacccgg tacggatcat tcccgatatc aattatcggc cggtgccggt caccccagac
     1681 ctcaatccgg tcaccgggcg gccgattgat cttagcttcg ccatcctgcg cagcgtctcg
     1741 ccctgccatt tggagttcat gcgcaacata ggcatgcacg gcacgatgtc gatctcgatt
     1801 ttgcgcggcg agcgactgtg gggattgatc gtttgccatc accgaacgcc gtactacgtc
     1861 gatctcgatg gccgccaagc ctgcgagcta gtcgcccagg ttctggcctg gcagatcggc
     1921 gtgatggaag agcgaccggt tcacgtggga agtggggcta caaacttctc actgctgaaa
     1981 caggccgggg atgtggagga aaacccagga ccacacgtga ctagtgtgag caagggcgag
     2041 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2101 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     2161 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     2221 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     2281 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     2341 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2401 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2461 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     2521 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2581 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2641 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2701 gccgccggga tcactctcgg catggacgag ctgtacaagt aagaattcga tatcaagctt
     2761 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2821 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2881 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2941 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3001 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3061 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3121 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3181 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3241 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3301 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     3361 taccgtcgac ccgggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     3421 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3481 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     3541 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     3601 aaatgtggta aaatcgataa ggatcttcct agagcatggc tacgtagata agtagcatgg
     3661 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg
     3721 cgctcgctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3781 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3841 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3901 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3961 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4021 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4081 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4141 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4201 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4261 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4321 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4381 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4441 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4501 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4561 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4621 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4681 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4741 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4801 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4861 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4921 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4981 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5041 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5101 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5161 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5221 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5281 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5341 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5401 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5461 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5521 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5581 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5641 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5701 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5761 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5821 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5881 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5941 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     6001 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     6061 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     6121 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctgcgc
     6181 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc
     6241 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
     6301 tccttgtagt taatgattaa cccgccatgc tact
//
