LOCUS       sequence-487074-        5554 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  Expresses human phosphomutant-NGN3 in mammalian cells directly fused
            to Dendra2 fluorescent protin plus FLAG and HA tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5554)
  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 5554)
  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-PM-NGN3-linker-3xFLAG-Dendra2-HA
FEATURES             Location/Qualifiers
     source          1..5554
                     /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             2463..2528
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2535..3221
                     /codon_start=1
                     /product="monomeric photoswitchable green-to-red 
                     fluorescent protein from Dendronephthya"
                     /label=Dendra2
                     /note="mammalian codon-optimized"
                     /translation="NTPGINLIKEDMRVKVHMEGNVNGHAFVIEGEGKGKPYEGTQTAN
                     LTVKEGAPLPFSYDILTTAVHYGNRVFTKYPEDIPDYFKQSFPEGYSWERTMTFEDKGI
                     CTIRSDISLEGDCFFQNVRFKGTNFPPNGPVMQKKTLKWEPSTEKLHVRDGLLVGNINM
                     ALLLEGGGHYLCDFKTTYKAKKVVQLPDAHFVDHRIEILGNDSDYNKVKLYEHAVARYS
                     PLPSQVW"
     CDS             3228..3254
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3258..3282)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3311..3432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3322..3341)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3376..3395
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3548..3565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3719..4307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3799..3818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4478..5269,5270..5338))
                     /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     5101..5120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5339..5443)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5511..5529
                     /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 caccatgacg cctcaaccct
     1801 cgggtgcgcc cactgtccaa gtgacccgtg agacggagcg gtccttcccc agagcctcgg
     1861 aagacgaagt gacctgcccc acgtccgccc cgcccagccc cactcgcaca cgggggaact
     1921 gcgcagaggc ggaagaggga ggctgccgag gggccccgag gaagctccgg gcacggcgcg
     1981 ggggacgcag ccggcctaag agcgagttgg cactgagcaa gcagcgacgg agtcggcgaa
     2041 agaaggccaa cgaccgcgag cgcaatcgaa tgcacaacct caactcggca ctggacgccc
     2101 tgcgcggtgt cctgcccacc ttcccagacg acgcgaagct caccaagatc gagacgctgc
     2161 gcttcgccca caactacatc tgggcgctga ctcaaacgct gcgcatagcg gaccacagct
     2221 tgtacgcgct ggagccgccg gcgccgcact gcggggagct gggcgcccca ggcggtgccc
     2281 ccggggactg ggggtccctc tacgccccag tctcccaggc tggcagcctg gctcccgccg
     2341 cgtcgctgga ggagaggccc gggctgctgg gggccacctt ttccgcctgc ttggccccag
     2401 gcagtctggc tttctcagat tttctgggct ccgctggctc cgctgctggt tctggcgaat
     2461 ttgactacaa agaccatgac ggtgattata aagatcatga catcgattac aaggatgacg
     2521 atgacaaggg taccaacacc ccgggaatta acctgatcaa ggaggacatg cgcgtgaagg
     2581 tgcacatgga gggcaacgtg aacggccacg ccttcgtgat cgagggcgag ggcaagggca
     2641 agccctacga gggcacccag accgccaacc tgaccgtgaa ggagggcgcc cccctgccct
     2701 tcagctacga catcctgacc accgccgtgc actacggcaa ccgggtgttc accaagtacc
     2761 ccgaggacat ccccgactac ttcaagcaga gcttccccga gggctacagc tgggagcgca
     2821 ccatgacctt cgaggacaag ggcatctgca ccatccgcag cgacatcagc ctggagggcg
     2881 actgcttctt ccagaacgtg cgcttcaagg gcaccaactt cccccccaac ggccccgtga
     2941 tgcagaagaa gaccctgaag tgggagccca gcaccgagaa gctgcacgtg cgcgacggcc
     3001 tgctggtggg caacatcaac atggccctgc tgctggaggg cggcggccac tacctgtgcg
     3061 acttcaagac cacctacaag gccaagaagg tggtgcagct gcccgacgcc cacttcgtgg
     3121 accaccgcat cgagatcctg ggcaacgaca gcgactacaa caaggtgaag ctgtacgagc
     3181 acgccgtggc ccgctacagc cccctgccca gccaggtgtg gggtacctac ccatacgatg
     3241 ttccagatta cgcttgaacc cagctttctt gtacaaagtg gtgatggccg gccgcttcga
     3301 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     3361 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     3421 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     3481 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtagcggccg cggcgctctt
     3541 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3601 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3661 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3721 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3781 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3841 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctctct tcgggaagcg
     3901 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3961 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4021 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4081 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4141 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4201 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4261 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4321 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4381 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4441 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4501 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4561 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4621 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4681 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4741 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4801 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4861 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4921 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4981 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5041 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5101 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5161 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5221 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5281 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5341 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5401 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5461 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     5521 tcacgaggcc ctttcgtcgg cgcgccgcgg ccgc
//
