LOCUS       sequence-463031-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of Ngn2x3HA with mRuby2 fluorescent reporter 
            with upstream open reading frame.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  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 7080)
  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: pMXs-uORFx3-mNgn2x3HA-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     117..133
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     protein_bind    222..246
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1169..1195
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1205..1231
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1241..1267
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1277..1333
                     /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             1334..2044
                     /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(2061..2085)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2102..2690
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2155..2175)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2573..2584)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2870..3339
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3469..3486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3640..4228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3720..3739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4392..5183,5184..5252))
                     /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     5015..5034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5253..5357)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5387..5980
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6043..6400
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6459..6875
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6805..6827
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6843..6859
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6885..7080,1..179)
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
ORIGIN
        1 tcggcgacac agtgtgggtc cgccgacacc agactaagaa cctagaacct cgctggaaag
       61 gaccttacac agtcctgctg accaccccca ccgccctcaa agtagacggc atcgcagctt
      121 ggatacacgc cgcccacgtg aaggctgccg accccggggg tggaccatcc tctagactgc
      181 cggatctagc tagttaatcg acggtatcga taagctatca aacaagtttg tacaaaaaag
      241 caggctccac catgggaacc aattcagtcg agtcgactgg atccaccatg ggttgaacca
      301 tgggttgaac catgggttga gatccggtac tactgccacc atgttcgtca aatctgagac
      361 tctggagttg aaggaggaag aggaggtact gatgctgctg ggctcggctt ccccggcctc
      421 ggcgaccctg accccgatgt cctccagcgc ggacgaggag gaggacgagg agctgcgccg
      481 gccgggctcc gcgcgtgggc agcgtggagc ggaagccggg cagggggtgc agggcagtcc
      541 ggcgtcgggt gccgggggtt gccggccagg gcggctgctg ggcctgatgc acgagtgcaa
      601 gcgtcgcccg tcgcgctcac gggccgtctc ccgaggtgcc aagacggcgg agacggtgca
      661 gcgcatcaag aagacccgca ggctcaaggc caacaaccgc gagcgcaacc gcatgcacaa
      721 cctaaacgcc gcgctggacg cgctgcgcga ggtgctgccc accttccccg aggatgccaa
      781 gctcacgaag atcgagacgc tgcgcttcgc ccacaattac atctgggcgc tcaccgagac
      841 tctgcgcctg gcggaccact gcgccggcgc cggtggcctc cagggggcgc tcttcacgga
      901 ggcggtgctc ctgagcccgg gagctgcgct cggcgccagc ggggacagcc cttctccacc
      961 ttcctcctgg agctgcacca acagcccggc gtcatcctcc aactccacgt ccccatacag
     1021 ctgcacttta tcgcccgcta gccccgggtc agacgtggac tactggcagc ccccacctcc
     1081 ggagaagcat cgttatgcgc ctcacctgcc cctcgccagg gactgtatcg ggtctggctc
     1141 cggatcaagt ggtggcagcg gatcaggata tccatacgat gtaccagact atgcgggatc
     1201 gggttacccc tacgacgtcc ccgactacgc cggctccggc tacccctacg acgtccctga
     1261 ttatgctgga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     1321 gaaccctgga cctatggtgt ctaagggcga agagctgatc aaggaaaata tgcgtatgaa
     1381 ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa tgcacaggtg aaggagaagg
     1441 caatccgtac atgggaactc aaaccatgag gatcaaagtc atcgagggag gacccctgcc
     1501 atttgccttt gacattcttg ccacgtcgtt catgtatggc agccgtactt ttatcaagta
     1561 cccgaaaggc attcctgatt tctttaaaca gtcctttcct gagggtttta cttgggaaag
     1621 agttacgaga tacgaagatg gtggagtcgt caccgtcatg caggacacca gccttgagga
     1681 tggctgtctc gtttaccacg tccaagtcag aggggtaaac tttccctcca atggtcccgt
     1741 gatgcagaag aagaccaagg gttgggagcc taatacagag atgatgtatc cagcagatgg
     1801 tggtctgagg ggatacactc atatggcact gaaagttgat ggtggtggcc atctgtcttg
     1861 ctctttcgta acaacttaca ggtcaaaaaa gaccgtcggg aacatcaaga tgcccggtat
     1921 ccatgccgtt gatcaccgcc tggaaaggtt agaggaaagt gacaatgaaa tgttcgtagt
     1981 acaacgcgaa cacgcagttg ccaagttcgc cgggcttggt ggtgggatgg acgagctgta
     2041 caagtaacgc tgatatctag acccagcttt cttgtacaaa gtggttcgat agcttatcga
     2101 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2161 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2221 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2281 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2341 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2401 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2461 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2521 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2581 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     2641 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     2701 tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     2761 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca
     2821 taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg
     2881 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga
     2941 acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg
     3001 ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag
     3061 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat
     3121 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     3181 caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat
     3241 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct
     3301 cctctgagtg attgactacc cgtcagcggg ggtctttcac atgcagcatg tatcaaaatt
     3361 aatttggttt tttttcttaa gtatttacat taaatggcca tagttgcatt aatgaatcgg
     3421 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3481 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3541 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3601 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3661 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3721 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3781 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3841 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3901 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3961 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4021 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4081 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4141 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4201 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4261 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4321 cttcacctag atccttttgc ggccggccgc aaatcaatct aaagtatata tgagtaaact
     4381 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4441 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4501 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4561 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4621 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4681 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4741 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4801 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4861 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4921 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4981 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5041 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5101 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5161 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5221 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5281 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5341 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgcataatg aaagacccca
     5401 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat
     5461 aactgagaat agaaaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc
     5521 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa
     5581 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     5641 caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga
     5701 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     5761 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     5821 aacccctcac tcggcgcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
     5881 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     5941 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
     6001 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc cagcaactta
     6061 tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
     6121 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tcggaacacc
     6181 cggccgcaac cctgggagac gtcccaggga cttcgggggc cgtttttgtg gcccgacctg
     6241 agtccaaaaa tcccgatcgt tttggactct ttggtgcacc cccctaatag gagggatatg
     6301 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     6361 cggtttggga ccgaagccgc gccgcgcgtc ttgtctgctg cagcatcgtt ctgtgttgtc
     6421 tctgtctgac tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct
     6481 taagtttgac cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag
     6541 atgtcaagaa gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg
     6601 gatggccgcg agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct
     6661 tttcacctgg cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct
     6721 tggcttttga cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc
     6781 ttcctccatc cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct
     6841 ccctttatcc agccctcact ccttctctag gcgcccccat atggccatat gagatcttat
     6901 atggggcacc cccgcccctt gtaaacttcc ctgaccctga catgacaaga gttactaaca
     6961 gcccctctct ccaagctcac ttacaggctc tctacttagt ccagcacgaa gtctggagac
     7021 ctctggcggc agcctaccaa gaacaactgg accgaccggt ggtacctcac ccttaccgag
//
