LOCUS       sequence-462998-        7068 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 7068)
  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 7068)
  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-uORFx2-mNgn2x3HA-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7068
                     /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             1157..1183
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1193..1219
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1229..1255
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1265..1321
                     /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             1322..2032
                     /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(2049..2073)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2090..2678
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2143..2163)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2561..2572)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2858..3327
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3457..3474)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3628..4216)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3708..3727)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4380..5171,5172..5240))
                     /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     5003..5022
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5241..5345)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5375..5968
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6031..6388
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6447..6863
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6793..6815
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6831..6847
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6873..7068,1..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 tgggttgaga tccggtacta ctgccaccat gttcgtcaaa tctgagactc tggagttgaa
      361 ggaggaagag gaggtactga tgctgctggg ctcggcttcc ccggcctcgg cgaccctgac
      421 cccgatgtcc tccagcgcgg acgaggagga ggacgaggag ctgcgccggc cgggctccgc
      481 gcgtgggcag cgtggagcgg aagccgggca gggggtgcag ggcagtccgg cgtcgggtgc
      541 cgggggttgc cggccagggc ggctgctggg cctgatgcac gagtgcaagc gtcgcccgtc
      601 gcgctcacgg gccgtctccc gaggtgccaa gacggcggag acggtgcagc gcatcaagaa
      661 gacccgcagg ctcaaggcca acaaccgcga gcgcaaccgc atgcacaacc taaacgccgc
      721 gctggacgcg ctgcgcgagg tgctgcccac cttccccgag gatgccaagc tcacgaagat
      781 cgagacgctg cgcttcgccc acaattacat ctgggcgctc accgagactc tgcgcctggc
      841 ggaccactgc gccggcgccg gtggcctcca gggggcgctc ttcacggagg cggtgctcct
      901 gagcccggga gctgcgctcg gcgccagcgg ggacagccct tctccacctt cctcctggag
      961 ctgcaccaac agcccggcgt catcctccaa ctccacgtcc ccatacagct gcactttatc
     1021 gcccgctagc cccgggtcag acgtggacta ctggcagccc ccacctccgg agaagcatcg
     1081 ttatgcgcct cacctgcccc tcgccaggga ctgtatcggg tctggctccg gatcaagtgg
     1141 tggcagcgga tcaggatatc catacgatgt accagactat gcgggatcgg gttaccccta
     1201 cgacgtcccc gactacgccg gctccggcta cccctacgac gtccctgatt atgctggaag
     1261 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     1321 tatggtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
     1381 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     1441 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     1501 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     1561 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     1621 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     1681 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     1741 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     1801 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     1861 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     1921 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     1981 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca agtaacgctg
     2041 atatctagac ccagctttct tgtacaaagt ggttcgatag cttatcgata atcaacctct
     2101 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2161 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2221 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2281 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2341 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2401 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2461 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     2521 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2581 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     2641 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacgataaaa
     2701 taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt
     2761 ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata
     2821 gagaagttca gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata
     2881 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat
     2941 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     3001 ggtccccaga tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg
     3061 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     3121 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     3181 cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct
     3241 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     3301 tgactacccg tcagcggggg tctttcacat gcagcatgta tcaaaattaa tttggttttt
     3361 tttcttaagt atttacatta aatggccata gttgcattaa tgaatcggcc aacgcgcggg
     3421 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3481 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3541 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3601 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3661 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3721 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3781 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3841 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3901 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3961 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4021 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4081 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4141 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4201 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4261 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4321 ccttttgcgg ccggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4381 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4441 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4501 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4561 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4621 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4681 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4741 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4801 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4861 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4921 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4981 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5041 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5101 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5161 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5221 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5281 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5341 ccgcgcacat ttccccgaaa agtgccacct gcataatgaa agaccccacc tgtaggtttg
     5401 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     5461 aaaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat
     5521 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg
     5581 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     5641 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     5701 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     5761 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     5821 ggcgcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     5881 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     5941 gactacccgt cagcgggggt ctttcatttg ggggctcgtc cgggatcggg agacccctgc
     6001 ccagggacca ccgacccacc accgggaggt aagctggcca gcaacttatc tgtgtctgtc
     6061 cgattgtcta gtgtctatga ctgattttat gcgcctgcgt cggtactagt tagctaacta
     6121 gctctgtatc tggcggaccc gtggtggaac tgacgagttc ggaacacccg gccgcaaccc
     6181 tgggagacgt cccagggact tcgggggccg tttttgtggc ccgacctgag tccaaaaatc
     6241 ccgatcgttt tggactcttt ggtgcacccc cctaatagga gggatatgtg gttctggtag
     6301 gagacgagaa cctaaaacag ttcccgcctc cgtctgaatt tttgctttcg gtttgggacc
     6361 gaagccgcgc cgcgcgtctt gtctgctgca gcatcgttct gtgttgtctc tgtctgactg
     6421 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     6481 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     6541 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     6601 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     6661 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     6721 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     6781 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     6841 ccctcactcc ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc
     6901 cgccccttgt aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc
     6961 aagctcactt acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag
     7021 cctaccaaga acaactggac cgaccggtgg tacctcaccc ttaccgag
//
