LOCUS       sequence-361612-        7682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector: CAG promoter driving expression of 
            nuclear envelope-tethered mKate2 (NLS-mKate2-EmerinTMD). Confers 
            hygromycin B resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7682)
  AUTHORS   Blin G, Sadurska D, Portero Migueles R, Chen N, Watson JA, Lowell S
  TITLE     Nessys: A new set of tools for the automated detection of nuclei 
            within intact tissues and dense 3D cultures.
  JOURNAL   PLoS Biol. 2019 Aug 9;17(8):e3000388. doi: 
            10.1371/journal.pbio.3000388. eCollection 2019 Aug.
  PUBMED    31398189
REFERENCE   2  (bases 1 to 7682)
  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: "PLoS 
            Biol."; date: "2019-08-9"; volume: "17(8):e3000388. doi"; pages: " 
            10.1371/journal.pbio.3000388. eCollection 2019 Aug"
COMMENT     Sequence Label: pPyCAG-NE-mKate2-IRES-Hph
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    309..443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        457..836
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1115..2131
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2055..2077
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2139..2158
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2204..2224
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2225..2917
                     /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"
     misc_feature    join(3117..3691,3692..3694,3695..3703)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3284..3301)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3511..3530
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3704..4729
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5032..5048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5032..5048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5045..5067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5056..5072
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5080..5086,5087..5104,5105..5110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5125..5146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5205..5400
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5251..5386
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5406..5540
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5608..5625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5779..6367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5859..5878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6538..7329,7330..7398))
                     /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     7161..7180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agctgttgtc atagtaatga ttgggtggaa acattccagg cctgggtgga gaggcttttt
       61 gcttcctctt gcaaaaccac actgacattc caggcctggg tggagaggct ttttgcttcc
      121 tcttgcaaaa ccacactgcc ctctggaggg cagttgccta gcaactaatt aaaagaggat
      181 gtcgcacggc cagctgcggt cagttagtca cttcctgctt aactgacttg acattttcta
      241 ttttaagagt cgggaggaaa attactgtgt tggaggccct ccgccatctt ctgaagctga
      301 atcgaattaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
      361 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
      421 atgtatctta tcatgtctgg atctgatatc atcgtcgaca ttgattattg actagttatt
      481 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      541 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      601 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      661 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      721 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      781 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
      841 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
      901 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggnsg
      961 cgcgccrggs ggggsggggs ggggsgrggg gsggggsggg gsgrggcgga gaggtgcggc
     1021 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     1081 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
     1141 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     1201 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
     1261 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
     1321 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
     1381 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     1441 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     1501 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1561 gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg
     1621 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     1681 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     1741 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     1801 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     1861 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     1921 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     1981 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     2041 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     2101 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     2161 gtctcatcat tttggcaaag aattcctcga ggccgccacc atgccaaaaa agaagagaaa
     2221 ggtagtgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
     2281 gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     2341 gaccatgaga atcaaggcgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     2401 taccagcttc atgtacggca gcaaaacctt catcaaccac acccagggca tccccgactt
     2461 ctttaagcag tccttccccg agggcttcac atgggagaga gtcaccacat acgaagacgg
     2521 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     2581 caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg
     2641 ctgggaggcc tccaccgaga ccctgtaccc cgctgacggc ggcctggaag gcagagccga
     2701 catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttgaaga ccacatacag
     2761 atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg acagaagact
     2821 ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc
     2881 cagatactgc gacctcccta gcaaactggg gcacagaggt agcggcagcg gtagccgtgc
     2941 tcctggggct gggctgggcc aggatcgcca ggtcccgctc tggggccagc tgctgctttt
     3001 cctggtcttt gtgatcgtcc tcttcttcat ttaccacttc atgcaggctg aagaaggcaa
     3061 ccccttctaa cctagggcgg ccgctcgata agcttgatat cgaattccgc cccccccccc
     3121 tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg
     3181 tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa
     3241 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg
     3301 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca
     3361 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc
     3421 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt
     3481 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg
     3541 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca
     3601 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac
     3661 gtggttttcc tttgaaaaac acgatgataa tatggccaca accatgaaaa agcctgaact
     3721 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     3781 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3841 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg ttcatcggca
     3901 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     3961 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     4021 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     4081 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     4141 gtggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     4201 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     4261 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     4321 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     4381 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4441 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4501 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4561 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4621 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4681 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagt cgagaaattg
     4741 atgatctatt aagcaataaa gacgtccact aaaatggaag tttttcctgt catactttgt
     4801 taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc tacgggggtg
     4861 ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta ctattgcttt
     4921 atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa ttaagggcca
     4981 gctcattcct ccactcacga tctatagatc cactagcttg gcgtaatcat ggtcatagct
     5041 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5101 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5161 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     5221 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     5281 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     5341 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     5401 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5461 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5521 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5581 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5641 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5701 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5761 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5821 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5881 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5941 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6001 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6061 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6181 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6421 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6481 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6661 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6841 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7021 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7381 gaaatgttga atactcatac tcttcctttt tcaatgaccg ctttggaaaa acaaagactg
     7441 tatttcctgg aaattaatgt ttattcaata aactgtgtat tcagctatat tcacatagtg
     7501 gtgaggctga aatgaggcgg gaagaggcgg ttggggctta attatatcaa tttgggtggc
     7561 cccacagcgc ctccaaggcg ccagtcctgt tttgacaagt tgcctctgga agcctctctt
     7621 ctttttctcc agagtaagcg gaggccaggg gcccccggcc tctgcttaat actaaaaaaa
     7681 ac
//
