LOCUS       sequence-462990-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of Isl1 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 7200)
  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 7200)
  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-uORFx3-Isl1-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7200
                     /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"
     regulatory      335..344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1131..1140
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1397..1453
                     /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             1454..2164
                     /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(2181..2205)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2222..2810
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2275..2295)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2693..2704)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2990..3459
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3589..3606)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3760..4348)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3840..3859)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4512..5303,5304..5372))
                     /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     5135..5154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5373..5477)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5507..6100
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6163..6520
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6579..6995
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6925..6947
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6963..6979
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(7005..7200,1..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 tgggttgaac catgggttga gatccggtac tactgccacc atgggagaca tgggcgatcc
      361 accaaaaaaa aaacgtctga tttccctgtg tgttggttgc ggcaatcaaa ttcacgacca
      421 gtatattctg agggtttctc cggatttgga gtggcatgca gcatgtttga aatgtgcgga
      481 gtgtaatcag tatttggacg aaagctgtac gtgctttgtt agggatggga aaacctactg
      541 taaaagagat tatatcaggt tgtacgggat caaatgcgcc aagtgcagca taggcttcag
      601 caagaacgac ttcgtgatgc gcgcccgctc taaggtgtac cacatcgagt gtttccgctg
      661 tgtagcctgc agccgacagc tcatcccggg agacgaattc gccctgcggg aggatgggct
      721 tttctgccgt gcagaccacg atgtggtgga gagagccagc ctgggagctg gagatcctct
      781 cagtcccttg catccagcgc ggcctctgca aatggcagcc gaacccatct cggctaggca
      841 gccagctctg cggccgcacg tccacaagca gccggagaag accacccgag tgcggactgt
      901 gctcaacgag aagcagctgc acaccttgcg gacctgctat gccgccaacc ctcggccaga
      961 tgcgctcatg aaggagcaac tagtggagat gacgggcctc agtcccagag tcatccgagt
     1021 gtggtttcaa aacaagcggt gcaaggacaa gaaacgcagc atcatgatga agcagctcca
     1081 gcagcagcaa cccaacgaca aaactaatat ccaggggatg acaggaactc ccatggtggc
     1141 tgctagtccg gagagacatg atggtggttt acaggctaac ccagtagagg tgcaaagtta
     1201 ccagccgccc tggaaagtac tgagtgactt cgccttgcaa agcgacatag atcagcctgc
     1261 ttttcagcaa ctggtcaatt tttcagaagg aggaccaggc tctaattcta ctggcagtga
     1321 agtagcatcg atgtcctcgc agctcccaga tacacccaac agcatggtag ccagtcctat
     1381 tgaggcagga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     1441 gaaccctgga cctatggtgt ctaagggcga agagctgatc aaggaaaata tgcgtatgaa
     1501 ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa tgcacaggtg aaggagaagg
     1561 caatccgtac atgggaactc aaaccatgag gatcaaagtc atcgagggag gacccctgcc
     1621 atttgccttt gacattcttg ccacgtcgtt catgtatggc agccgtactt ttatcaagta
     1681 cccgaaaggc attcctgatt tctttaaaca gtcctttcct gagggtttta cttgggaaag
     1741 agttacgaga tacgaagatg gtggagtcgt caccgtcatg caggacacca gccttgagga
     1801 tggctgtctc gtttaccacg tccaagtcag aggggtaaac tttccctcca atggtcccgt
     1861 gatgcagaag aagaccaagg gttgggagcc taatacagag atgatgtatc cagcagatgg
     1921 tggtctgagg ggatacactc atatggcact gaaagttgat ggtggtggcc atctgtcttg
     1981 ctctttcgta acaacttaca ggtcaaaaaa gaccgtcggg aacatcaaga tgcccggtat
     2041 ccatgccgtt gatcaccgcc tggaaaggtt agaggaaagt gacaatgaaa tgttcgtagt
     2101 acaacgcgaa cacgcagttg ccaagttcgc cgggcttggt ggtgggatgg acgagctgta
     2161 caagtaacgc tgatatctag acccagcttt cttgtacaaa gtggttcgat agcttatcga
     2221 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2281 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2341 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2401 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2461 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2521 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2581 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2641 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2701 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     2761 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     2821 tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     2881 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca
     2941 taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg
     3001 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga
     3061 acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg
     3121 ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag
     3181 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat
     3241 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     3301 caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat
     3361 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct
     3421 cctctgagtg attgactacc cgtcagcggg ggtctttcac atgcagcatg tatcaaaatt
     3481 aatttggttt tttttcttaa gtatttacat taaatggcca tagttgcatt aatgaatcgg
     3541 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3601 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3661 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3721 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3781 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3841 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3901 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3961 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4021 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4081 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4141 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4201 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4261 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4321 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4381 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4441 cttcacctag atccttttgc ggccggccgc aaatcaatct aaagtatata tgagtaaact
     4501 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4561 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4621 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4681 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4741 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4801 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4861 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4921 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4981 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5041 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5101 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5161 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5221 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5281 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5341 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5401 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5461 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgcataatg aaagacccca
     5521 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat
     5581 aactgagaat agaaaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc
     5641 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa
     5701 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     5761 caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga
     5821 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     5881 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     5941 aacccctcac tcggcgcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
     6001 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     6061 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
     6121 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc cagcaactta
     6181 tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
     6241 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tcggaacacc
     6301 cggccgcaac cctgggagac gtcccaggga cttcgggggc cgtttttgtg gcccgacctg
     6361 agtccaaaaa tcccgatcgt tttggactct ttggtgcacc cccctaatag gagggatatg
     6421 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     6481 cggtttggga ccgaagccgc gccgcgcgtc ttgtctgctg cagcatcgtt ctgtgttgtc
     6541 tctgtctgac tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct
     6601 taagtttgac cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag
     6661 atgtcaagaa gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg
     6721 gatggccgcg agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct
     6781 tttcacctgg cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct
     6841 tggcttttga cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc
     6901 ttcctccatc cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct
     6961 ccctttatcc agccctcact ccttctctag gcgcccccat atggccatat gagatcttat
     7021 atggggcacc cccgcccctt gtaaacttcc ctgaccctga catgacaaga gttactaaca
     7081 gcccctctct ccaagctcac ttacaggctc tctacttagt ccagcacgaa gtctggagac
     7141 ctctggcggc agcctaccaa gaacaactgg accgaccggt ggtacctcac ccttaccgag
//
