LOCUS       sequence-487072-        7131 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  Expresses human 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 7131)
  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 7131)
  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-NGN3-linker-3xFLAG-Dendra2-HA
FEATURES             Location/Qualifiers
     source          1..7131
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    144..164
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        168..547
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        549..826
                     /label=chicken beta-actin promoter
     intron          827..1843
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1767..1789
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1851..1870
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1897..1921
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2606..2671
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2678..3364
                     /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             3371..3397
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3401..3425)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(3426..4000,4001..4003,4004..4012)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3593..3610)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3820..3839
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4013..4612
                     /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(4013..4032)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4509..4529
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(4613..4633)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    4661..4782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4672..4691)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4726..4745
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4898..4915)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5069..5657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5149..5168)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5828..6619,6620..6688))
                     /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     6451..6470
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6689..6793)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    6905..6925
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
       61 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
      121 tttcgtcggc gcgccgcggc cgccaacttt gtatagaaaa gttgctcgac attgattatt
      181 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      241 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      301 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      361 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      421 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      481 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      541 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      601 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      661 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
      721 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      781 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      841 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      901 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      961 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1021 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
     1081 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
     1141 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
     1201 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
     1261 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
     1321 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
     1381 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
     1441 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
     1501 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
     1561 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
     1621 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
     1681 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
     1741 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
     1801 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
     1861 ggttattgtg ctgtctcatc attttggcaa agaattgcaa gtttgtacaa aaaagcaggc
     1921 tgccaccatg acgcctcaac cctcgggtgc gcccactgtc caagtgaccc gtgagacgga
     1981 gcggtccttc cccagagcct cggaagacga agtgacctgc cccacgtccg ccccgcccag
     2041 ccccactcgc acacggggga actgcgcaga ggcggaagag ggaggctgcc gaggggcccc
     2101 gaggaagctc cgggcacggc gcgggggacg cagccggcct aagagcgagt tggcactgag
     2161 caagcagcga cggagtcggc gaaagaaggc caacgaccgc gagcgcaatc gaatgcacaa
     2221 cctcaactcg gcactggacg ccctgcgcgg tgtcctgccc accttcccag acgacgcgaa
     2281 gctcaccaag atcgagacgc tgcgcttcgc ccacaactac atctgggcgc tgactcaaac
     2341 gctgcgcata gcggaccaca gcttgtacgc gctggagccg ccggcgccgc actgcgggga
     2401 gctgggcagc ccaggcggtt cccccgggga ctgggggtcc ctctactccc cagtctccca
     2461 ggctggcagc ctgagtcccg ccgcgtcgct ggaggagcga cccgggctgc tgggggccac
     2521 cttttccgcc tgcttgagcc caggcagtct ggctttctca gattttctgg gctccgctgg
     2581 ctccgctgct ggttctggcg aatttgacta caaagaccat gacggtgatt ataaagatca
     2641 tgacatcgat tacaaggatg acgatgacaa ggatatcaac accccgggaa ttaacctgat
     2701 caaggaggac atgcgcgtga aggtgcacat ggagggcaac gtgaacggcc acgccttcgt
     2761 gatcgagggc gagggcaagg gcaagcccta cgagggcacc cagaccgcca acctgaccgt
     2821 gaaggagggc gcccccctgc ccttcagcta cgacatcctg accaccgccg tgcactacgg
     2881 caaccgggtg ttcaccaagt accccgagga catccccgac tacttcaagc agagcttccc
     2941 cgagggctac agctgggagc gcaccatgac cttcgaggac aagggcatct gcaccatccg
     3001 cagcgacatc agcctggagg gcgactgctt cttccagaac gtgcgcttca agggcaccaa
     3061 cttccccccc aacggccccg tgatgcagaa gaagaccctg aagtgggagc ccagcaccga
     3121 gaagctgcac gtgcgcgacg gcctgctggt gggcaacatc aacatggccc tgctgctgga
     3181 gggcggcggc cactacctgt gcgacttcaa gaccacctac aaggccaaga aggtggtgca
     3241 gctgcccgac gcccacttcg tggaccaccg catcgagatc ctgggcaacg acagcgacta
     3301 caacaaggtg aagctgtacg agcacgccgt ggcccgctac agccccctgc ccagccaggt
     3361 gtgggatatc tacccatacg atgttccaga ttacgcttga acccagcttt cttgtacaaa
     3421 gtgggcccct ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     3481 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     3541 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     3601 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     3661 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     3721 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     3781 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     3841 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     3901 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     3961 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatgaccga
     4021 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     4081 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     4141 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     4201 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     4261 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     4321 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     4381 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     4441 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     4501 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     4561 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacaacttta
     4621 ttatacatag ttgatggccg gccgcttcga gcagacatga taagatacat tgatgagttt
     4681 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4741 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4801 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     4861 tacaaatgtg gtagcggccg cggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4921 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4981 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5221 acctgtccgc ctttctctct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5701 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5761 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5821 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5881 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5941 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6001 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6061 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6121 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6181 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6241 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6301 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6361 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6421 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6481 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6541 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6601 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6661 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6721 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6781 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6841 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtcgg cgcgccgcgg
     6901 ccgccaactt tgtatagaaa agttgctcga cattgattat tgactagtta ttaatagtaa
     6961 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     7021 gtaaatggcc mtrcsgcatc rgmrymrrwk swrmttgtac tgagagtgca ccatagggga
     7081 tcgggagatc tcccgatccg tcgacgtcag gtggcacttt tcggggaaat g
//
