LOCUS       sequence-463013-        7056 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 7056)
  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 7056)
  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-uORFx1-mNgn2x3HA-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7056
                     /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             1145..1171
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1181..1207
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1217..1243
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1253..1309
                     /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             1310..2020
                     /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(2037..2061)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2078..2666
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2131..2151)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2549..2560)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2846..3315
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3445..3462)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3616..4204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3696..3715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4368..5159,5160..5228))
                     /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     4991..5010
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5229..5333)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5363..5956
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6019..6376
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6435..6851
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6781..6803
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6819..6835
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6861..7056,1..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 ggttgagatc
      301 cggtactact gccaccatgt tcgtcaaatc tgagactctg gagttgaagg aggaagagga
      361 ggtactgatg ctgctgggct cggcttcccc ggcctcggcg accctgaccc cgatgtcctc
      421 cagcgcggac gaggaggagg acgaggagct gcgccggccg ggctccgcgc gtgggcagcg
      481 tggagcggaa gccgggcagg gggtgcaggg cagtccggcg tcgggtgccg ggggttgccg
      541 gccagggcgg ctgctgggcc tgatgcacga gtgcaagcgt cgcccgtcgc gctcacgggc
      601 cgtctcccga ggtgccaaga cggcggagac ggtgcagcgc atcaagaaga cccgcaggct
      661 caaggccaac aaccgcgagc gcaaccgcat gcacaaccta aacgccgcgc tggacgcgct
      721 gcgcgaggtg ctgcccacct tccccgagga tgccaagctc acgaagatcg agacgctgcg
      781 cttcgcccac aattacatct gggcgctcac cgagactctg cgcctggcgg accactgcgc
      841 cggcgccggt ggcctccagg gggcgctctt cacggaggcg gtgctcctga gcccgggagc
      901 tgcgctcggc gccagcgggg acagcccttc tccaccttcc tcctggagct gcaccaacag
      961 cccggcgtca tcctccaact ccacgtcccc atacagctgc actttatcgc ccgctagccc
     1021 cgggtcagac gtggactact ggcagccccc acctccggag aagcatcgtt atgcgcctca
     1081 cctgcccctc gccagggact gtatcgggtc tggctccgga tcaagtggtg gcagcggatc
     1141 aggatatcca tacgatgtac cagactatgc gggatcgggt tacccctacg acgtccccga
     1201 ctacgccggc tccggctacc cctacgacgt ccctgattat gctggaagcg gagctactaa
     1261 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta tggtgtctaa
     1321 gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa
     1381 cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg gaactcaaac
     1441 catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca ttcttgccac
     1501 gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc ctgatttctt
     1561 taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg aagatggtgg
     1621 agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt accacgtcca
     1681 agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga ccaagggttg
     1741 ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat acactcatat
     1801 ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa cttacaggtc
     1861 aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc accgcctgga
     1921 aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg cagttgccaa
     1981 gttcgccggg cttggtggtg ggatggacga gctgtacaag taacgctgat atctagaccc
     2041 agctttcttg tacaaagtgg ttcgatagct tatcgataat caacctctgg attacaaaat
     2101 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     2161 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     2221 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     2281 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     2341 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     2401 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     2461 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     2521 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     2581 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     2641 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cgataaaata aaagatttta
     2701 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     2761 taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga
     2821 tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     2881 agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg
     2941 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     3001 cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     3061 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     3121 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt
     3181 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
     3241 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
     3301 agcgggggtc tttcacatgc agcatgtatc aaaattaatt tggttttttt tcttaagtat
     3361 ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3421 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3481 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3541 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3601 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3661 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3721 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3781 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3841 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3901 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3961 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4021 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4081 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4141 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4201 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4261 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttgcggcc
     4321 ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4381 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4441 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4501 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4561 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4621 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4681 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4741 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4801 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4861 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4921 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4981 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5041 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5101 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5161 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5221 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5281 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5341 ccccgaaaag tgccacctgc ataatgaaag accccacctg taggtttggc aagctagctt
     5401 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat
     5461 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca
     5521 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga
     5581 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     5641 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     5701 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     5761 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     5821 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     5881 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     5941 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc
     6001 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
     6061 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg
     6121 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc
     6181 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg
     6241 gactctttgg tgcacccccc taataggagg gatatgtggt tctggtagga gacgagaacc
     6301 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg
     6361 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     6421 tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta ggtcactgga
     6481 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta
     6541 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta
     6601 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc
     6661 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg
     6721 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc
     6781 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt
     6841 ctctaggcgc ccccatatgg ccatatgaga tcttatatgg ggcacccccg ccccttgtaa
     6901 acttccctga ccctgacatg acaagagtta ctaacagccc ctctctccaa gctcacttac
     6961 aggctctcta cttagtccag cacgaagtct ggagacctct ggcggcagcc taccaagaac
     7021 aactggaccg accggtggta cctcaccctt accgag
//
