LOCUS       sequence-480849-        6363 bp    DNA     circular SYN 28-JUL-2025
DEFINITION  Expresses NES-mBaoJin and mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6363)
  AUTHORS   Zhang H, Lesnov GD, Subach OM, Zhang W, Kuzmicheva TP, Vlaskina AV, 
            Samygina VR, Chen L, Ye X, Nikolaeva AY, Gabdulkhakov A, Papadaki S,
            Qin W, Borshchevskiy V, Perfilov MM, Gavrikov AS, Drobizhev M, 
            Mishin AS, Piatkevich KD, Subach FV
  TITLE     Bright and stable monomeric green fluorescent protein derived from 
            StayGold.
  JOURNAL   Nat Methods. 2024 Apr;21(4):657-665. doi: 
            10.1038/s41592-024-02203-y. Epub 2024 Feb 26.
  PUBMED    38409224
REFERENCE   2  (bases 1 to 6363)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02203-y"; journalName: "Nat Methods"; date: 
            "2024-04"; volume: "21"; issue: "4"; pages: "657-665"
COMMENT     Sequence Label: pAAV-AscI-CAG-NES-mBaoJin-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..6363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     primer_bind     3533..3555
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3617..3636
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3684..3710
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             3720..4421
                     /codon_start=1
                     /product="bright and photostable monomeric green 
                     fluorescent protein derived from StayGold (Zhang et al., 
                     2024)"
                     /label=mBaoJin
                     /label=mStayGold
                     /note="/vntifkey=21"
                     /translation="MVSKGEEENMASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTTFGYGMKYYTKYPSGLKNWFREVMPGGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVPQIPRDDGVECPVTL
                     LYPLLSDKSKYVEAHQYTICKPLHNQPAPDVPYHWIRKQYTQSKDDAEERDHICQSETL
                     EAHLKGMDELYK"
     CDS             4452..4508
                     /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             4542..5252
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4687..4705)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4955..4974
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    5265..5853
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5318..5338)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5736..5747)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5896..6017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5907..5926)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5961..5980
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6179..6308
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagtcgtag gttaattaag gcgcgccgcc taggccattg acgtcaataa tgacgtatgt
     2701 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2761 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2821 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2881 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     2941 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     3001 tttttaatta ttttgtgcag cgatgggggc gggggggggg gggggggcgc gccmcggggg
     3061 ggsggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggsssmgg
     3121 nngggggggg gsgggssggg gsgwggcggw gaggtgcggc ggcagccaat cagagcggcg
     3181 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     3241 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
     3301 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     3361 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
     3421 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
     3481 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
     3541 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
     3601 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
     3661 ttggatccga caccatgctt caacttcctc ctcttgaacg tcttactctt tcgagatcta
     3721 tggtctcaaa gggagaggag gaaaacatgg ctagtacacc atttaaattt caacttaaag
     3781 gaaccatcaa tggcaaatcg tttaccgttg aaggcgaagg tgaagggaac tcacatgaag
     3841 gttctcataa aggaaaatat gtttgtacaa gtggaaaact accgatgtca tgggcagcac
     3901 ttgggacaac ctttggttat ggaatgaaat attataccaa atatcctagt ggactgaaga
     3961 actggtttcg tgaagtaatg cccggaggct ttacctacga tcgtcatatt caatataaag
     4021 gcgatgggag tatccatgca aaacaccaac actttatgaa aaatgggact tatcacaaca
     4081 ttgtagaatt taccggtcag gattttaaag aaaatagtcc agtcttaact ggagatatga
     4141 atgtctcatt accgaatgaa gtcccacaaa tacccagaga tgatggagta gaatgcccag
     4201 tgaccttgct ttatccttta ttatcggata aatcaaaata cgtcgaggct caccaatata
     4261 caatctgcaa gcctcttcat aatcaaccag cacctgatgt cccatatcac tggattcgta
     4321 aacaatacac acaaagcaaa gatgatgccg aggaacgcga tcatatctgt caatcagaga
     4381 ctctcgaagc acacttaaag ggcatggacg agctgtataa ggaattccga ccggttcacg
     4441 tgggaagtgg ggctacaaac ttctcactgc tgaaacaggc cggggatgtg gaggaaaacc
     4501 caggaccaca cgtgactagt ggtggttctg gttctggtac catggtgagc aagggcgagg
     4561 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     4621 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     4681 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     4741 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     4801 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     4861 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     4921 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     4981 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     5041 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     5101 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     5161 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     5221 ccaccggcgg catggacgag ctgtataagt aaaagcttat cgataatcaa cctctggatt
     5281 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     5341 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     5401 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     5461 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     5521 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     5581 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     5641 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     5701 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     5761 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     5821 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc
     5881 ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     5941 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     6001 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     6061 agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg
     6121 atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag
     6181 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     6241 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     6301 gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6361 gct
//
