LOCUS       sequence-361595-        7594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for tagBFP-3xNLS from the CAG promoter. 
            Confers hygromycin B resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7594)
  AUTHORS   Malaguti M, Migueles RP, Annoh J, Sadurska D, Blin G, Lowell S
  TITLE     SyNPL: Synthetic notch pluripotent cell lines to monitor and 
            manipulate cell interactions in vitro and in vivo.
  JOURNAL   Development. 2022 May 26. pii: 275525. doi: 10.1242/dev.200226.
  PUBMED    35616331
REFERENCE   2  (bases 1 to 7594)
  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: 
            "Development. 2022 May 26. pii: 275525. doi: 10.1242/dev.200226."
COMMENT     Sequence Label: pPyCAG-tagBFP-3xNLS-IRES-Hph
FEATURES             Location/Qualifiers
     source          1..7594
                     /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"
     promoter        838..1113
                     /label=chicken beta-actin promoter
     intron          1114..2130
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2054..2076
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2138..2157
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2200..2898
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2914..2934
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2938..2958
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2962..2982
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    join(3029..3603,3604..3606,3607..3615)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3196..3213)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3423..3442
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3616..4641
                     /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(4944..4960)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4944..4960)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4957..4979)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4968..4984
                     /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(4992..4998,4999..5016,5017..5022))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5037..5058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5117..5312
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5163..5298
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5318..5452
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5520..5537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5691..6279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5771..5790)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6450..7241,7242..7310))
                     /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     7073..7092
                     /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 gggggggggc
      961 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
     1021 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1081 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     1141 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     1201 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
     1261 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
     1321 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
     1381 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1441 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc
     1501 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg
     1561 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc
     1621 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg
     1681 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg
     1741 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc
     1801 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt
     1861 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg
     1921 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     1981 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     2041 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     2101 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     2161 tctcatcatt ttggcaaaga attcctcgag gccgccacca tgagcgagct gattaaggag
     2221 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     2281 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     2341 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     2401 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     2461 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     2521 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     2581 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     2641 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     2701 agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc
     2761 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     2821 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     2881 ctggggcaca agcttaatgg cgccacaggt agtccaaaaa agaagagaaa ggtagatcca
     2941 aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg tattataagc ggccgctcga
     3001 taagcttgat atcgaattcc gccccccccc cctctccctc ccccccccct aacgttactg
     3061 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     3121 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     3181 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     3241 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     3301 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     3361 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     3421 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     3481 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa
     3541 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     3601 aatatggcca caaccatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     3661 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     3721 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     3781 ggtttctaca aagatcgtta tgttcatcgg cactttgcat cggccgcgct cccgattccg
     3841 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     3901 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     3961 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     4021 ttcggaccgc aaggaatcgg tcaatacact acgtggcgtg atttcatatg cgcgattgct
     4081 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     4141 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     4201 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     4261 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     4321 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     4381 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     4441 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     4501 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     4561 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     4621 cgtccgaggg caaaggaata gtcgagaaat tgatgatcta ttaagcaata aagacgtcca
     4681 ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca
     4741 ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct
     4801 gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat
     4861 aatttaaaca agcaaaacca aattaagggc cagctcattc ctccactcac gatctataga
     4921 tccactagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4981 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5041 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5101 tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     5161 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5221 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5281 gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa
     5341 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     5401 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat
     5461 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5521 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5581 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5641 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5701 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5761 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5821 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5881 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5941 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6001 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6061 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6121 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6181 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6241 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6301 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6361 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6421 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6481 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6541 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6601 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6661 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6721 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6781 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6841 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6901 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6961 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7021 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7081 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7141 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7201 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7261 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7321 tttcaatgac cgctttggaa aaacaaagac tgtatttcct ggaaattaat gtttattcaa
     7381 taaactgtgt attcagctat attcacatag tggtgaggct gaaatgaggc gggaagaggc
     7441 ggttggggct taattatatc aatttgggtg gccccacagc gcctccaagg cgccagtcct
     7501 gttttgacaa gttgcctctg gaagcctctc ttctttttct ccagagtaag cggaggccag
     7561 gggcccccgg cctctgctta atactaaaaa aaac
//
