LOCUS       sequence-463024-        8633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of Ngn2x3HA, Isl1, and Lhx3 for motor neuron 
            reprogramming.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8633)
  AUTHORS   Wang NB, Adewumi HO, Lende-Dorn BA, Beitz AM, O'Shea TM, Galloway KE
  TITLE     Compact transcription factor cassettes generate functional, 
            engraftable motor neurons by direct conversion.
  JOURNAL   Cell Syst. 2025 Mar 10:101206. doi: 10.1016/j.cels.2025.101206.
  PUBMED    40086435
REFERENCE   2  (bases 1 to 8633)
  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. 
            2025 Mar 10:101206. doi: 10.1016/j.cels.2025.101206."
COMMENT     Sequence Label: pMXs-NIL (with mNgn2x3HA)
FEATURES             Location/Qualifiers
     source          1..8633
                     /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"
     regulatory      2078..2087
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2344..2397
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    complement(3614..3638)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3655..4243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3708..3728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4126..4137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4423..4892
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5022..5039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5193..5781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5273..5292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5945..6736,6737..6805))
                     /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     6568..6587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6806..6910)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             6940..7533
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    7596..7953
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             8012..8428
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8358..8380
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     8396..8412
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(8438..8633,1..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 ggagacatgg gcgatccacc aaaaaaaaaa
     1321 cgtctgattt ccctgtgtgt tggttgcggc aatcaaattc acgaccagta tattctgagg
     1381 gtttctccgg atttggagtg gcatgcagca tgtttgaaat gtgcggagtg taatcagtat
     1441 ttggacgaaa gctgtacgtg ctttgttagg gatgggaaaa cctactgtaa aagagattat
     1501 atcaggttgt acgggatcaa atgcgccaag tgcagcatag gcttcagcaa gaacgacttc
     1561 gtgatgcgcg cccgctctaa ggtgtaccac atcgagtgtt tccgctgtgt agcctgcagc
     1621 cgacagctca tcccgggaga cgaattcgcc ctgcgggagg atgggctttt ctgccgtgca
     1681 gaccacgatg tggtggagag agccagcctg ggagctggag atcctctcag tcccttgcat
     1741 ccagcgcggc ctctgcaaat ggcagccgaa cccatctcgg ctaggcagcc agctctgcgg
     1801 ccgcacgtcc acaagcagcc ggagaagacc acccgagtgc ggactgtgct caacgagaag
     1861 cagctgcaca ccttgcggac ctgctatgcc gccaaccctc ggccagatgc gctcatgaag
     1921 gagcaactag tggagatgac gggcctcagt cccagagtca tccgagtgtg gtttcaaaac
     1981 aagcggtgca aggacaagaa acgcagcatc atgatgaagc agctccagca gcagcaaccc
     2041 aacgacaaaa ctaatatcca ggggatgaca ggaactccca tggtggctgc tagtccggag
     2101 agacatgatg gtggtttaca ggctaaccca gtagaggtgc aaagttacca gccgccctgg
     2161 aaagtactga gtgacttcgc cttgcaaagc gacatagatc agcctgcttt tcagcaactg
     2221 gtcaattttt cagaaggagg accaggctct aattctactg gcagtgaagt agcatcgatg
     2281 tcctcgcagc tcccagatac acccaacagc atggtagcca gtcctattga ggcaggcagt
     2341 ggtgagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg
     2401 ctgctagaag cagaactcga ttgccaccga gagaggcccg gtgcccctgg agcttctgcc
     2461 ctctgtacct tcagcaggac tccagagatc ccgatgtgtg caggctgtga ccagcacatc
     2521 ttggaccgtt tcatccttaa ggctctggac cgacattggc acagcaagtg tctcaagtgc
     2581 agtgactgcc acgtccctct ggctgagcgc tgcttcagcc gcggggagag cgtctactgc
     2641 aaagacgact tctttaagcg cttcgggacc aagtgcgccg catgccagct gggcatcccg
     2701 cccacgcagg tggtgcgccg cgcccaggac ttcgtgtacc acctgcattg cttcgcctgc
     2761 gtggtctgca agcggcagct ggccacgggc gacgagttct acctcatgga agacagccgg
     2821 ctggtgtgca aggcggacta cgaaacagcc aagcagcgag aagccgaggc cacagccaag
     2881 cggccgcgca ccaccatcac cgccaagcag ctggagacgc tgaagagcgc ctacaacact
     2941 tcgcccaagc cggcgcgcca cgtgcgcgag cagctctcct ccgagactgg cctggacatg
     3001 cgagtggtgc aggtgtggtt ccagaatcgc cgggctaagg aaaagagact gaagaaagac
     3061 gctggccggc agcgctgggg acagtatttc cgcaatatga agcgctcccg cggcagttcc
     3121 aagtccgaca aggacagcat ccaggaggga caagacagcg acgccgaagt ctccttcact
     3181 gatgagccgt ccatggctga catggggcct gctaatggcc tgtacagcag cctgggagag
     3241 cctgcccctg cgttgggccg gcccgtagga ggcctgggca gctttaccct ggatcacgga
     3301 ggcttgacgg gtccagagca gtaccgagag ctacgcccag gcagccccta tggcatcccc
     3361 ccatctcctg cagcccccca gagccttcct ggcccccagc ctctcctctc cagcctggta
     3421 tacccagaca ccaacttgag ccttgtccct tcagggcccc caggtggacc cccacccatg
     3481 agggtgctgg ctggaaatgg gcccagctcc gacctgtcca cagagagcag ttctggctac
     3541 ccagactttc ctgctagccc tgcttcctgg ctggatgaag tagaccatgc tcagttctaa
     3601 cgctgatatc tagacccagc tttcttgtac aaagtggttc gatagcttat cgataatcaa
     3661 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3721 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3781 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3841 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3901 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3961 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     4021 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     4081 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     4141 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     4201 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacga
     4261 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     4321 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga
     4381 gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca
     4441 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg
     4501 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     4561 cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc
     4621 cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg
     4681 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc
     4741 tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa
     4801 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
     4861 gtgattgact acccgtcagc gggggtcttt cacatgcagc atgtatcaaa attaatttgg
     4921 ttttttttct taagtattta cattaaatgg ccatagttgc attaatgaat cggccaacgc
     4981 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5041 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5101 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5161 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5221 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5281 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5341 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5401 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5461 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5521 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5581 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5641 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5701 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5761 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5821 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5881 tagatccttt tgcggccggc cgcaaatcaa tctaaagtat atatgagtaa acttggtctg
     5941 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6001 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6061 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6121 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6181 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6241 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6301 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6361 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6421 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6481 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6541 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6601 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6661 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6721 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6781 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6841 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6901 gggttccgcg cacatttccc cgaaaagtgc cacctgcata atgaaagacc ccacctgtag
     6961 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata cataactgag
     7021 aatagaaaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg ggccaaacag
     7081 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga
     7141 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     7201 agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc
     7261 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     7321 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     7381 cactcggcgc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa
     7441 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag
     7501 tgattgacta cccgtcagcg ggggtctttc atttgggggc tcgtccggga tcgggagacc
     7561 cctgcccagg gaccaccgac ccaccaccgg gaggtaagct ggccagcaac ttatctgtgt
     7621 ctgtccgatt gtctagtgtc tatgactgat tttatgcgcc tgcgtcggta ctagttagct
     7681 aactagctct gtatctggcg gacccgtggt ggaactgacg agttcggaac acccggccgc
     7741 aaccctggga gacgtcccag ggacttcggg ggccgttttt gtggcccgac ctgagtccaa
     7801 aaatcccgat cgttttggac tctttggtgc acccccctaa taggagggat atgtggttct
     7861 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
     7921 ggaccgaagc cgcgccgcgc gtcttgtctg ctgcagcatc gttctgtgtt gtctctgtct
     7981 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     8041 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     8101 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc
     8161 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc
     8221 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt
     8281 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc
     8341 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta
     8401 tccagccctc actccttctc taggcgcccc catatggcca tatgagatct tatatggggc
     8461 acccccgccc cttgtaaact tccctgaccc tgacatgaca agagttacta acagcccctc
     8521 tctccaagct cacttacagg ctctctactt agtccagcac gaagtctgga gacctctggc
     8581 ggcagcctac caagaacaac tggaccgacc ggtggtacct cacccttacc gag
//
