LOCUS       sequence-441130-        5423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of a single-cell discriminating version of 
            mKate2 fluorescent protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5423)
  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 5423)
  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-mKate2
FEATURES             Location/Qualifiers
     source          1..5423
                     /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             1514..2209
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     primer_bind     complement(2285..2305)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2687..2827
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2687..2816
                     /label=AAV2 ITR
     rep_origin      2902..3357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2989..3008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3199..3220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3374..3393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3493..3515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3553..3571)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3639..3743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3744..3812,3813..4604)
                     /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(3962..3981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4775..5362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5263..5282
                     /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 gagccaccat
     1441 gaaaagggtg gctccccaga agcagatgag catagcaaag aaaaagaagg tgagaaggcg
     1501 gcggcgccgt cgagtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
     1561 gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga
     1621 gggcacccag accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga
     1681 catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat
     1741 ccccgacttc tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata
     1801 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     1861 ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa
     1921 aacactcggc tgggaggcct ccaccgagac cctgtacccc gctgacggcg gcctggaagg
     1981 cagagccgac atggccctga agctcgtggg cgggggccac ctgatctgca acttgaagac
     2041 cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga
     2101 cagaagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc agcacgaggt
     2161 ggctgtggcc agatactgcg acctccctag caaactgggg cacagatgag atatcgaatt
     2221 ccgctcgaga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2281 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2341 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggtta gttcttgcca
     2401 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     2461 ctgacaattc cgtggtgttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     2521 tctagcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     2581 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     2641 tgggaggttt tttaaagcgg ccgccacgtg cggaccgagc ggccgcagga acccctagtg
     2701 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     2761 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     2821 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2881 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2941 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     3001 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3061 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     3121 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3181 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     3241 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     3301 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     3361 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3421 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3481 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3541 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3601 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3661 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3721 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3781 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3841 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3901 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3961 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4021 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4081 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4141 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4201 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4261 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4321 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4381 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4441 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4501 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4561 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4621 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4681 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4741 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4801 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4861 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4921 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4981 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5041 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5101 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5161 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5221 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5281 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5341 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5401 ttgctggcct tttgctcaca tgt
//
