LOCUS       sequence-463004-        7126 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 7126)
  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 7126)
  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-uORFx6-mNgn2x3HA-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7126
                     /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             1215..1241
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1251..1277
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1287..1313
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1323..1379
                     /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             1380..2090
                     /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(2107..2131)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2148..2736
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2201..2221)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2619..2630)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2916..3385
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3515..3532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3686..4274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3766..3785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4438..5229,5230..5298))
                     /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     5061..5080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5299..5403)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5433..6026
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6089..6446
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6505..6921
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6851..6873
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6889..6905
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6931..7126,1..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 agtcgagtcg actggatcca ccatgggttg
      301 aaccatgggt tgaaccatgg gttgagatcc accatgggtt gaaccatggg ttgaaccatg
      361 ggttgagatc cggtactact gccaccatgt tcgtcaaatc tgagactctg gagttgaagg
      421 aggaagagga ggtactgatg ctgctgggct cggcttcccc ggcctcggcg accctgaccc
      481 cgatgtcctc cagcgcggac gaggaggagg acgaggagct gcgccggccg ggctccgcgc
      541 gtgggcagcg tggagcggaa gccgggcagg gggtgcaggg cagtccggcg tcgggtgccg
      601 ggggttgccg gccagggcgg ctgctgggcc tgatgcacga gtgcaagcgt cgcccgtcgc
      661 gctcacgggc cgtctcccga ggtgccaaga cggcggagac ggtgcagcgc atcaagaaga
      721 cccgcaggct caaggccaac aaccgcgagc gcaaccgcat gcacaaccta aacgccgcgc
      781 tggacgcgct gcgcgaggtg ctgcccacct tccccgagga tgccaagctc acgaagatcg
      841 agacgctgcg cttcgcccac aattacatct gggcgctcac cgagactctg cgcctggcgg
      901 accactgcgc cggcgccggt ggcctccagg gggcgctctt cacggaggcg gtgctcctga
      961 gcccgggagc tgcgctcggc gccagcgggg acagcccttc tccaccttcc tcctggagct
     1021 gcaccaacag cccggcgtca tcctccaact ccacgtcccc atacagctgc actttatcgc
     1081 ccgctagccc cgggtcagac gtggactact ggcagccccc acctccggag aagcatcgtt
     1141 atgcgcctca cctgcccctc gccagggact gtatcgggtc tggctccgga tcaagtggtg
     1201 gcagcggatc aggatatcca tacgatgtac cagactatgc gggatcgggt tacccctacg
     1261 acgtccccga ctacgccggc tccggctacc cctacgacgt ccctgattat gctggaagcg
     1321 gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta
     1381 tggtgtctaa gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag
     1441 gttcggtcaa cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg
     1501 gaactcaaac catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca
     1561 ttcttgccac gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc
     1621 ctgatttctt taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg
     1681 aagatggtgg agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt
     1741 accacgtcca agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga
     1801 ccaagggttg ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat
     1861 acactcatat ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa
     1921 cttacaggtc aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc
     1981 accgcctgga aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg
     2041 cagttgccaa gttcgccggg cttggtggtg ggatggacga gctgtacaag taacgctgat
     2101 atctagaccc agctttcttg tacaaagtgg ttcgatagct tatcgataat caacctctgg
     2161 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     2221 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     2281 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     2341 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     2401 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     2461 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     2521 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     2581 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     2641 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     2701 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cgataaaata
     2761 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     2821 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     2881 gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc
     2941 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg
     3001 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     3061 tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     3121 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     3181 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     3241 gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct
     3301 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     3361 actacccgtc agcgggggtc tttcacatgc agcatgtatc aaaattaatt tggttttttt
     3421 tcttaagtat ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga
     3481 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3541 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3601 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3661 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3721 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3781 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3841 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3901 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3961 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4021 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4081 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4141 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4201 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4261 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4321 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4381 ttttgcggcc ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4441 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4501 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4561 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4621 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4681 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4741 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4801 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4861 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4921 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4981 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5041 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5101 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5161 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5221 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5281 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5341 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5401 gcgcacattt ccccgaaaag tgccacctgc ataatgaaag accccacctg taggtttggc
     5461 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa
     5521 aagttcagat caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct
     5581 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg
     5641 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt
     5701 ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc
     5761 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
     5821 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
     5881 cgcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt
     5941 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
     6001 ctacccgtca gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc
     6061 agggaccacc gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg
     6121 attgtctagt gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc
     6181 tctgtatctg gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg
     6241 ggagacgtcc cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc
     6301 gatcgttttg gactctttgg tgcacccccc taataggagg gatatgtggt tctggtagga
     6361 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga
     6421 agccgcgccg cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg
     6481 tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta
     6541 ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga
     6601 cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac
     6661 ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg
     6721 catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc
     6781 cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc
     6841 ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc
     6901 ctcactcctt ctctaggcgc ccccatatgg ccatatgaga tcttatatgg ggcacccccg
     6961 ccccttgtaa acttccctga ccctgacatg acaagagtta ctaacagccc ctctctccaa
     7021 gctcacttac aggctctcta cttagtccag cacgaagtct ggagacctct ggcggcagcc
     7081 taccaagaac aactggaccg accggtggta cctcaccctt accgag
//
