LOCUS       sequence-441206-        5684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of a single-cell discriminating version of 
            miRFP670 fluorescent protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5684)
  AUTHORS   Szelenyi ER, Navarrete JS, Murry AD, Zhang Y, Girven KS, Kuo L, 
            Cline MM, Bernstein MX, Burdyniuk M, Bowler B, Goodwin NL, Juarez B,
            Zweifel LS, Golden SA
  TITLE     An arginine-rich nuclear localization signal (ArgiNLS) strategy for 
            streamlined image segmentation of single cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Aug 6;121(32):e2320250121. doi: 
            10.1073/pnas.2320250121. Epub 2024 Jul 29.
  PUBMED    39074275
REFERENCE   2  (bases 1 to 5684)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-08-6"; volume: "121(32):e2320250121. 
            doi"; pages: " 10.1073/pnas.2320250121. Epub 2024 Jul 29"
COMMENT     Sequence Label: pAAV-EF1a-ArgiNLS-miRFP670
FEATURES             Location/Qualifiers
     source          1..5684
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1520..2464
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="VAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDHR
                     VIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEY
                     CGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTGY
                     DRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVTY
                     QPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLVV
                     CHHYLPRFIRFELRAICKRLAERIATRITALES"
     primer_bind     complement(2546..2566)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2948..3088
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2948..3077
                     /label=AAV2 ITR
     rep_origin      3163..3618
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3250..3269)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3460..3481
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3635..3654)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3754..3776
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3814..3832)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3900..4004
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4005..4073,4074..4865)
                     /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(4223..4242)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5036..5623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5524..5543
                     /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 gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccgtacc ggatcctcta gaggtaccgc
     1441 caccatgaaa agggtggctc cccagaagca gatgagcata gcaaagaaaa agaaggtgag
     1501 aaggcggcgg cgccgtcgag tagcaggtca tgcctctggc agccccgcat tcgggaccgc
     1561 ctctcattcg aattgcgaac atgaagagat ccacctcgcc ggctcgatcc agccgcatgg
     1621 cgcgcttctg gtcgtcagcg aacatgatca tcgcgtcatc caggccagcg ccaacgccgc
     1681 ggaatttctg aatctcggaa gcgtactcgg cgttccgctc gccgagatcg acggcgatct
     1741 gttgatcaag atcctgccgc atctcgatcc caccgccgaa ggcatgccgg tcgcggtgcg
     1801 ctgccggatc ggcaatccct ctacggagta ctgcggtctg atgcatcggc ctccggaagg
     1861 cgggctgatc atcgaactcg aacgtgccgg cccgtcgatc gatctgtcag gcacgctggc
     1921 gccggcgctg gagcggatcc gcacggcggg ttcactgcgc gcgctgtgcg atgacaccgt
     1981 gctgctgttt cagcagtgca ccggctacga ccgggtgatg gtgtatcgtt tcgatgagca
     2041 aggccacggc ctggtattct ccgagtgcca tgtgcctggg ctcgaatcct atttcggcaa
     2101 ccgctatccg tcgtcgactg tcccgcagat ggcgcggcag ctgtacgtgc ggcagcgcgt
     2161 ccgcgtgctg gtcgacgtca cctatcagcc ggtgccgctg gagccgcggc tgtcgccgct
     2221 gaccgggcgc gatctcgaca tgtcgggctg cttcctgcgc tcgatgtcgc cgtgccatct
     2281 gcagttcctg aaggacatgg gcgtgcgcgc caccctggcg gtgtcgctgg tggtcggcgg
     2341 caagctgtgg ggcctggttg tctgtcacca ttatctgccg cgcttcatcc gtttcgagct
     2401 gcgggcgatc tgcaaacggc tcgccgaaag gatcgcgacg cggatcaccg cgcttgagag
     2461 ctaactcgag gatatcgaat tccgctcgag ataatcaacc tctggattac aaaatttgtg
     2521 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     2581 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     2641 aatcctggtt agttcttgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     2701 caggggctcg gctgttgggc actgacaatt ccgtggtgtt tatttgtgaa atttgtgatg
     2761 ctattgcttt atttgtaacc atctagcttt atttgtgaaa tttgtgatgc tattgcttta
     2821 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     2881 tttcaggttc agggggagat gtgggaggtt ttttaaagcg gccgccacgt gcggaccgag
     2941 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     3001 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     3061 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     3121 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     3181 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     3241 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3301 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3361 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3421 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3481 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     3541 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3601 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3661 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3721 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3781 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3841 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3901 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3961 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4021 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4081 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4141 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4201 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4261 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4321 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4381 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4441 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4501 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4561 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4621 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4681 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4741 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4801 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4861 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4921 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4981 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5041 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5101 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5161 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5221 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5281 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5341 agcggtcggg ctgaacgggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5401 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5461 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5521 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5581 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5641 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
