LOCUS       sequence-487094-        6038 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  Expressed a nuclear mScarleti fluorophoro in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6038)
  AUTHORS   Miller A, Biga V, Rowntree A, Chhatriwala M, Woods F, Marinopoulou 
            E, Kamath A, Vallier L, Papalopulu N
  TITLE     NGN3 oscillatory expression controls the timing of human pancreatic 
            endocrine differentiation
  JOURNAL   bioRxiv 2024.01.10.574974; doi: 10.1101/2024.01.10.574974.
REFERENCE   2  (bases 1 to 6038)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.01.10.574974; doi: 10.1101/2024.01.10.574974."
COMMENT     Sequence Label: CAG-NLS-mScarleti-NLS-IRES-Puromycin
FEATURES             Location/Qualifiers
     source          1..6038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        406..683
                     /label=chicken beta-actin promoter
     intron          684..1700
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1624..1646
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1708..1727
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1754..1778
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1788..1808
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1809..2504
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1942..1960)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2210..2229
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2505..2531
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     protein_bind    complement(2535..2559)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(2560..3134,3135..3137,3138..3146)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2727..2744)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2954..2973
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3147..3746
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3147..3166)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3643..3663
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(3747..3767)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3795..3916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3806..3825)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3860..3879
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4032..4049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4203..4791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4283..4302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4962..5753,5754..5822))
                     /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     5585..5604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5823..5927)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5995..6013
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 caactttgta tagaaaagtt gctcgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
      421 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
      481 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
      541 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      601 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      661 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
      721 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      781 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
      841 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
      901 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
      961 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     1021 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     1081 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     1141 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     1201 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     1261 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     1321 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     1381 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     1441 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     1501 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     1561 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     1621 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     1681 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     1741 ttggcaaaga attgcaagtt tgtacaaaaa agcaggctgc caccatgcca aaaaagaaga
     1801 gaaaggtaat ggtgagcaag ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc
     1861 acatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1921 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttct
     1981 cctgggacat cctgtcccct cagttcatgt acggctccag ggccttcatc aagcaccccg
     2041 ccgacatccc cgactactat aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga
     2101 tgaacttcga ggacggcggc gccgtgaccg tgacccagga cacctccctg gaggacggca
     2161 ccctgatcta caaggtgaag ctccgcggca ccaacttccc tcctgacggc cccgtaatgc
     2221 agaagaagac aatgggctgg gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc
     2281 tgaagggcga cattaagatg gccctgcgcc tgaaggacgg cggccgctac ctggcggact
     2341 tcaagaccac ctacaaggcc aagaagcccg tgcagatgcc cggcgcctac aacgtcgacc
     2401 gcaagttgga catcacctcc cacaacgagg actacaccgt ggtggaacag tacgaacgct
     2461 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagcctgct gccaagaggg
     2521 tcaagttgga ctagacccag ctttcttgta caaagtgggc ccctctccct cccccccccc
     2581 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     2641 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     2701 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     2761 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     2821 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     2881 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     2941 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     3001 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     3061 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     3121 aacacgatga taatatggcc acaaccatga ccgagtacaa gcccacggtg cgcctcgcca
     3181 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     3241 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3301 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3361 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3421 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3481 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     3541 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3601 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     3661 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3721 gcatgacccg caagcccggt gcctgacaac tttattatac atagttgatg gccggccgct
     3781 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3841 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3901 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3961 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtagcg gccgcggcgc
     4021 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4081 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct ctcttcggga
     4381 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4621 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4861 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4921 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4981 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5041 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5101 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5161 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5221 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5281 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5341 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5401 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5461 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5521 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5581 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5641 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5701 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5761 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5821 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5881 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5941 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     6001 cgtatcacga ggccctttcg tcggcgcgcc gcggccgc
//
