LOCUS       sequence-463015-        7034 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 7034)
  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 7034)
  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-mNgn2x3HA-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7034
                     /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             1123..1149
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1159..1185
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1195..1221
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1231..1287
                     /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             1288..1998
                     /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(2015..2039)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2056..2644
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2109..2129)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2527..2538)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2824..3293
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3423..3440)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3594..4182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3674..3693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4346..5137,5138..5206))
                     /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     4969..4988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5207..5311)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5341..5934
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    5997..6354
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6413..6829
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6759..6781
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6797..6813
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6839..7034,1..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 actggatccg gtactactgc caccatgttc
      301 gtcaaatctg agactctgga gttgaaggag gaagaggagg tactgatgct gctgggctcg
      361 gcttccccgg cctcggcgac cctgaccccg atgtcctcca gcgcggacga ggaggaggac
      421 gaggagctgc gccggccggg ctccgcgcgt gggcagcgtg gagcggaagc cgggcagggg
      481 gtgcagggca gtccggcgtc gggtgccggg ggttgccggc cagggcggct gctgggcctg
      541 atgcacgagt gcaagcgtcg cccgtcgcgc tcacgggccg tctcccgagg tgccaagacg
      601 gcggagacgg tgcagcgcat caagaagacc cgcaggctca aggccaacaa ccgcgagcgc
      661 aaccgcatgc acaacctaaa cgccgcgctg gacgcgctgc gcgaggtgct gcccaccttc
      721 cccgaggatg ccaagctcac gaagatcgag acgctgcgct tcgcccacaa ttacatctgg
      781 gcgctcaccg agactctgcg cctggcggac cactgcgccg gcgccggtgg cctccagggg
      841 gcgctcttca cggaggcggt gctcctgagc ccgggagctg cgctcggcgc cagcggggac
      901 agcccttctc caccttcctc ctggagctgc accaacagcc cggcgtcatc ctccaactcc
      961 acgtccccat acagctgcac tttatcgccc gctagccccg ggtcagacgt ggactactgg
     1021 cagcccccac ctccggagaa gcatcgttat gcgcctcacc tgcccctcgc cagggactgt
     1081 atcgggtctg gctccggatc aagtggtggc agcggatcag gatatccata cgatgtacca
     1141 gactatgcgg gatcgggtta cccctacgac gtccccgact acgccggctc cggctacccc
     1201 tacgacgtcc ctgattatgc tggaagcgga gctactaact tcagcctgct gaagcaggct
     1261 ggagacgtgg aggagaaccc tggacctatg gtgtctaagg gcgaagagct gatcaaggaa
     1321 aatatgcgta tgaaggtggt catggaaggt tcggtcaacg gccaccaatt caaatgcaca
     1381 ggtgaaggag aaggcaatcc gtacatggga actcaaacca tgaggatcaa agtcatcgag
     1441 ggaggacccc tgccatttgc ctttgacatt cttgccacgt cgttcatgta tggcagccgt
     1501 acttttatca agtacccgaa aggcattcct gatttcttta aacagtcctt tcctgagggt
     1561 tttacttggg aaagagttac gagatacgaa gatggtggag tcgtcaccgt catgcaggac
     1621 accagccttg aggatggctg tctcgtttac cacgtccaag tcagaggggt aaactttccc
     1681 tccaatggtc ccgtgatgca gaagaagacc aagggttggg agcctaatac agagatgatg
     1741 tatccagcag atggtggtct gaggggatac actcatatgg cactgaaagt tgatggtggt
     1801 ggccatctgt cttgctcttt cgtaacaact tacaggtcaa aaaagaccgt cgggaacatc
     1861 aagatgcccg gtatccatgc cgttgatcac cgcctggaaa ggttagagga aagtgacaat
     1921 gaaatgttcg tagtacaacg cgaacacgca gttgccaagt tcgccgggct tggtggtggg
     1981 atggacgagc tgtacaagta acgctgatat ctagacccag ctttcttgta caaagtggtt
     2041 cgatagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     2101 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     2161 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     2221 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     2281 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     2341 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     2401 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     2461 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     2521 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     2581 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     2641 ccgcatcgat accgtcgacg ataaaataaa agattttatt tagtctccag aaaaaggggg
     2701 gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg
     2761 catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa
     2821 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     2881 caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct
     2941 gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttc
     3001 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
     3061 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
     3121 aataaaagag cccacaaccc ctcactcggg gcgccagtcc tccgattgac tgagtcgccc
     3181 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
     3241 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcacatgcag
     3301 catgtatcaa aattaatttg gttttttttc ttaagtattt acattaaatg gccatagttg
     3361 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3421 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3481 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3541 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3601 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3661 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3721 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3781 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3841 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3901 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3961 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4021 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4081 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4141 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4201 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4261 ttatcaaaaa ggatcttcac ctagatcctt ttgcggccgg ccgcaaatca atctaaagta
     4321 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4381 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4441 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4501 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4561 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4621 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4681 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4741 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4801 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4861 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4921 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4981 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5041 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5101 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5161 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5221 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5281 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgcat
     5341 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
     5401 atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac
     5461 agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
     5521 aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg
     5581 ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct
     5641 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt
     5701 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
     5761 ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact gagtcgcccg
     5821 ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc
     5881 cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg
     5941 ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc
     6001 tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc
     6061 ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac
     6121 gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt
     6181 tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg caccccccta
     6241 ataggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc
     6301 tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat
     6361 cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt agggccagac
     6421 tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc ggatcgctca
     6481 caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg cagaatggcc
     6541 aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca tcacccaggt
     6601 taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc cctacatcgt
     6661 gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg tacaccctaa
     6721 gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc ctcgttcgac
     6781 cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccc ccatatggcc
     6841 atatgagatc ttatatgggg cacccccgcc ccttgtaaac ttccctgacc ctgacatgac
     6901 aagagttact aacagcccct ctctccaagc tcacttacag gctctctact tagtccagca
     6961 cgaagtctgg agacctctgg cggcagccta ccaagaacaa ctggaccgac cggtggtacc
     7021 tcacccttac cgag
//
