LOCUS       sequence-463823-        3997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of Ngn2x3HA with mRuby2 fluorescent reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3997)
  AUTHORS   Wang NB, Lende-Dorn BA, Beitz AM, Han P, Adewumi HO, O'Shea TM, 
            Galloway KE
  TITLE     Proliferation history and transcription factor levels drive direct 
            conversion to motor neurons.
  JOURNAL   Cell Syst. 2025 Apr 16;16(4):101205. doi: 
            10.1016/j.cels.2025.101205. Epub 2025 Mar 13.
  PUBMED    40086434
REFERENCE   2  (bases 1 to 3997)
  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: "Cell 
            Syst."; date: "2025-04-16"; volume: "16(4):101205. doi"; pages: " 
            10.1016/j.cels.2025.101205. Epub 2025 Mar 13"
COMMENT     Sequence Label: pENTR-mNgn2x3HA-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..3997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      104..190
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      282..309
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     317..340
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    359..458
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1335..1361
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1371..1397
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1407..1433
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1443..1499
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1500..2210
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     protein_bind    complement(2227..2326)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2438..2457)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2449..3258
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2516..2535)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3348..3936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3837..3856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg
       61 gggacgtcta actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc
      121 tcagtcggaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
      181 taggacaaat ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg
      241 ggcaggacgc ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga
      301 tggccttttt gcgtttctac aaactcttcc tgttagttag ttacttaagc tcgggcccca
      361 aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg ataagcaatg
      421 cttttttata atgccaactt tgtacaaaaa agcaggctcc accatgggaa ccaattcagt
      481 cgactggatc cggtactact gccaccatgt tcgtcaaatc tgagactctg gagttgaagg
      541 aggaagagga ggtactgatg ctgctgggct cggcttcccc ggcctcggcg accctgaccc
      601 cgatgtcctc cagcgcggac gaggaggagg acgaggagct gcgccggccg ggctccgcgc
      661 gtgggcagcg tggagcggaa gccgggcagg gggtgcaggg cagtccggcg tcgggtgccg
      721 ggggttgccg gccagggcgg ctgctgggcc tgatgcacga gtgcaagcgt cgcccgtcgc
      781 gctcacgggc cgtctcccga ggtgccaaga cggcggagac ggtgcagcgc atcaagaaga
      841 cccgcaggct caaggccaac aaccgcgagc gcaaccgcat gcacaaccta aacgccgcgc
      901 tggacgcgct gcgcgaggtg ctgcccacct tccccgagga tgccaagctc acgaagatcg
      961 agacgctgcg cttcgcccac aattacatct gggcgctcac cgagactctg cgcctggcgg
     1021 accactgcgc cggcgccggt ggcctccagg gggcgctctt cacggaggcg gtgctcctga
     1081 gcccgggagc tgcgctcggc gccagcgggg acagcccttc tccaccttcc tcctggagct
     1141 gcaccaacag cccggcgtca tcctccaact ccacgtcccc atacagctgc actttatcgc
     1201 ccgctagccc cgggtcagac gtggactact ggcagccccc acctccggag aagcatcgtt
     1261 atgcgcctca cctgcccctc gccagggact gtatcgggtc tggctccgga tcaagtggtg
     1321 gcagcggatc aggatatcca tacgatgtac cagactatgc gggatcgggt tacccctacg
     1381 acgtccccga ctacgccggc tccggctacc cctacgacgt ccctgattat gctggaagcg
     1441 gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta
     1501 tggtgtctaa gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag
     1561 gttcggtcaa cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg
     1621 gaactcaaac catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca
     1681 ttcttgccac gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc
     1741 ctgatttctt taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg
     1801 aagatggtgg agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt
     1861 accacgtcca agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga
     1921 ccaagggttg ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat
     1981 acactcatat ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa
     2041 cttacaggtc aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc
     2101 accgcctgga aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg
     2161 cagttgccaa gttcgccggg cttggtggtg ggatggacga gctgtacaag taacgctgat
     2221 atctagaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     2281 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag
     2341 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     2401 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     2461 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     2521 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     2581 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     2641 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     2701 tttatccgta ctcctggtga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     2761 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     2821 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     2881 tttcgtctag ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     2941 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     3001 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     3061 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     3121 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     3181 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     3241 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacattattc agattgggcc
     3301 ccgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3361 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3421 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3481 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3541 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3601 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3661 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3721 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3781 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3841 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3901 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3961 cggttcctgg ccttttgctg gccttttgct cacatgt
//
