LOCUS       sequence-462993-        7176 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 7176)
  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 7176)
  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-uORFx1-Isl1-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7176
                     /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      311..320
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1107..1116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1373..1429
                     /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             1430..2140
                     /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(2157..2181)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2198..2786
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2251..2271)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2669..2680)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2966..3435
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3565..3582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3736..4324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3816..3835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4488..5279,5280..5348))
                     /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     5111..5130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5349..5453)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5483..6076
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6139..6496
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6555..6971
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6901..6923
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6939..6955
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(6981..7176,1..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 ggttgagatc
      301 cggtactact gccaccatgg gagacatggg cgatccacca aaaaaaaaac gtctgatttc
      361 cctgtgtgtt ggttgcggca atcaaattca cgaccagtat attctgaggg tttctccgga
      421 tttggagtgg catgcagcat gtttgaaatg tgcggagtgt aatcagtatt tggacgaaag
      481 ctgtacgtgc tttgttaggg atgggaaaac ctactgtaaa agagattata tcaggttgta
      541 cgggatcaaa tgcgccaagt gcagcatagg cttcagcaag aacgacttcg tgatgcgcgc
      601 ccgctctaag gtgtaccaca tcgagtgttt ccgctgtgta gcctgcagcc gacagctcat
      661 cccgggagac gaattcgccc tgcgggagga tgggcttttc tgccgtgcag accacgatgt
      721 ggtggagaga gccagcctgg gagctggaga tcctctcagt cccttgcatc cagcgcggcc
      781 tctgcaaatg gcagccgaac ccatctcggc taggcagcca gctctgcggc cgcacgtcca
      841 caagcagccg gagaagacca cccgagtgcg gactgtgctc aacgagaagc agctgcacac
      901 cttgcggacc tgctatgccg ccaaccctcg gccagatgcg ctcatgaagg agcaactagt
      961 ggagatgacg ggcctcagtc ccagagtcat ccgagtgtgg tttcaaaaca agcggtgcaa
     1021 ggacaagaaa cgcagcatca tgatgaagca gctccagcag cagcaaccca acgacaaaac
     1081 taatatccag gggatgacag gaactcccat ggtggctgct agtccggaga gacatgatgg
     1141 tggtttacag gctaacccag tagaggtgca aagttaccag ccgccctgga aagtactgag
     1201 tgacttcgcc ttgcaaagcg acatagatca gcctgctttt cagcaactgg tcaatttttc
     1261 agaaggagga ccaggctcta attctactgg cagtgaagta gcatcgatgt cctcgcagct
     1321 cccagataca cccaacagca tggtagccag tcctattgag gcaggaagcg gagctactaa
     1381 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta tggtgtctaa
     1441 gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa
     1501 cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg gaactcaaac
     1561 catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca ttcttgccac
     1621 gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc ctgatttctt
     1681 taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg aagatggtgg
     1741 agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt accacgtcca
     1801 agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga ccaagggttg
     1861 ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat acactcatat
     1921 ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa cttacaggtc
     1981 aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc accgcctgga
     2041 aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg cagttgccaa
     2101 gttcgccggg cttggtggtg ggatggacga gctgtacaag taacgctgat atctagaccc
     2161 agctttcttg tacaaagtgg ttcgatagct tatcgataat caacctctgg attacaaaat
     2221 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     2281 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     2341 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     2401 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     2461 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     2521 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     2581 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     2641 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     2701 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     2761 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cgataaaata aaagatttta
     2821 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     2881 taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga
     2941 tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     3001 agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg
     3061 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     3121 cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     3181 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     3241 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt
     3301 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
     3361 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
     3421 agcgggggtc tttcacatgc agcatgtatc aaaattaatt tggttttttt tcttaagtat
     3481 ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3541 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3601 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3661 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3721 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3781 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3841 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3901 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3961 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4021 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4081 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4141 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4201 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4261 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4321 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4381 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttgcggcc
     4441 ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4501 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4561 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4621 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4681 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4741 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4801 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4861 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4921 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4981 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5041 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5101 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5161 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5221 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5281 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5341 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5401 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5461 ccccgaaaag tgccacctgc ataatgaaag accccacctg taggtttggc aagctagctt
     5521 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat
     5581 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca
     5641 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga
     5701 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     5761 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     5821 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     5881 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     5941 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     6001 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     6061 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc
     6121 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
     6181 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg
     6241 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc
     6301 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg
     6361 gactctttgg tgcacccccc taataggagg gatatgtggt tctggtagga gacgagaacc
     6421 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg
     6481 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     6541 tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta ggtcactgga
     6601 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta
     6661 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta
     6721 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc
     6781 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg
     6841 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc
     6901 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt
     6961 ctctaggcgc ccccatatgg ccatatgaga tcttatatgg ggcacccccg ccccttgtaa
     7021 acttccctga ccctgacatg acaagagtta ctaacagccc ctctctccaa gctcacttac
     7081 aggctctcta cttagtccag cacgaagtct ggagacctct ggcggcagcc taccaagaac
     7141 aactggaccg accggtggta cctcaccctt accgag
//
