LOCUS       sequence-463032-        7188 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 7188)
  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 7188)
  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-uORFx2-Isl1-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7188
                     /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      323..332
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1119..1128
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1385..1441
                     /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             1442..2152
                     /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(2169..2193)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2210..2798
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2263..2283)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2681..2692)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2978..3447
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3577..3594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3748..4336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3828..3847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4500..5291,5292..5360))
                     /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     5123..5142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5361..5465)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5495..6088
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6151..6508
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6567..6983
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6913..6935
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6951..6967
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6993..7188,1..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 tgggttgaga tccggtacta ctgccaccat gggagacatg ggcgatccac caaaaaaaaa
      361 acgtctgatt tccctgtgtg ttggttgcgg caatcaaatt cacgaccagt atattctgag
      421 ggtttctccg gatttggagt ggcatgcagc atgtttgaaa tgtgcggagt gtaatcagta
      481 tttggacgaa agctgtacgt gctttgttag ggatgggaaa acctactgta aaagagatta
      541 tatcaggttg tacgggatca aatgcgccaa gtgcagcata ggcttcagca agaacgactt
      601 cgtgatgcgc gcccgctcta aggtgtacca catcgagtgt ttccgctgtg tagcctgcag
      661 ccgacagctc atcccgggag acgaattcgc cctgcgggag gatgggcttt tctgccgtgc
      721 agaccacgat gtggtggaga gagccagcct gggagctgga gatcctctca gtcccttgca
      781 tccagcgcgg cctctgcaaa tggcagccga acccatctcg gctaggcagc cagctctgcg
      841 gccgcacgtc cacaagcagc cggagaagac cacccgagtg cggactgtgc tcaacgagaa
      901 gcagctgcac accttgcgga cctgctatgc cgccaaccct cggccagatg cgctcatgaa
      961 ggagcaacta gtggagatga cgggcctcag tcccagagtc atccgagtgt ggtttcaaaa
     1021 caagcggtgc aaggacaaga aacgcagcat catgatgaag cagctccagc agcagcaacc
     1081 caacgacaaa actaatatcc aggggatgac aggaactccc atggtggctg ctagtccgga
     1141 gagacatgat ggtggtttac aggctaaccc agtagaggtg caaagttacc agccgccctg
     1201 gaaagtactg agtgacttcg ccttgcaaag cgacatagat cagcctgctt ttcagcaact
     1261 ggtcaatttt tcagaaggag gaccaggctc taattctact ggcagtgaag tagcatcgat
     1321 gtcctcgcag ctcccagata cacccaacag catggtagcc agtcctattg aggcaggaag
     1381 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     1441 tatggtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
     1501 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     1561 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     1621 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     1681 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     1741 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     1801 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     1861 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     1921 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     1981 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     2041 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     2101 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca agtaacgctg
     2161 atatctagac ccagctttct tgtacaaagt ggttcgatag cttatcgata atcaacctct
     2221 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2281 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2341 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2401 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2461 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2521 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2581 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     2641 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2701 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     2761 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacgataaaa
     2821 taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt
     2881 ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata
     2941 gagaagttca gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata
     3001 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat
     3061 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     3121 ggtccccaga tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg
     3181 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     3241 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     3301 cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct
     3361 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     3421 tgactacccg tcagcggggg tctttcacat gcagcatgta tcaaaattaa tttggttttt
     3481 tttcttaagt atttacatta aatggccata gttgcattaa tgaatcggcc aacgcgcggg
     3541 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3601 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3661 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3721 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3781 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3841 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3901 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3961 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4021 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4081 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4141 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4201 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4261 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4321 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4381 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4441 ccttttgcgg ccggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4501 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4561 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4621 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4681 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4741 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4801 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4861 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4921 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4981 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5041 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5101 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5161 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5221 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5281 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5341 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5401 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5461 ccgcgcacat ttccccgaaa agtgccacct gcataatgaa agaccccacc tgtaggtttg
     5521 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     5581 aaaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat
     5641 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg
     5701 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     5761 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     5821 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     5881 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     5941 ggcgcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     6001 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     6061 gactacccgt cagcgggggt ctttcatttg ggggctcgtc cgggatcggg agacccctgc
     6121 ccagggacca ccgacccacc accgggaggt aagctggcca gcaacttatc tgtgtctgtc
     6181 cgattgtcta gtgtctatga ctgattttat gcgcctgcgt cggtactagt tagctaacta
     6241 gctctgtatc tggcggaccc gtggtggaac tgacgagttc ggaacacccg gccgcaaccc
     6301 tgggagacgt cccagggact tcgggggccg tttttgtggc ccgacctgag tccaaaaatc
     6361 ccgatcgttt tggactcttt ggtgcacccc cctaatagga gggatatgtg gttctggtag
     6421 gagacgagaa cctaaaacag ttcccgcctc cgtctgaatt tttgctttcg gtttgggacc
     6481 gaagccgcgc cgcgcgtctt gtctgctgca gcatcgttct gtgttgtctc tgtctgactg
     6541 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     6601 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     6661 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     6721 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     6781 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     6841 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     6901 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     6961 ccctcactcc ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc
     7021 cgccccttgt aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc
     7081 aagctcactt acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag
     7141 cctaccaaga acaactggac cgaccggtgg tacctcaccc ttaccgag
//
