LOCUS       sequence-465030-        7246 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 7246)
  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 7246)
  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-uORFx6-Isl1-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7246
                     /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      381..390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1177..1186
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1443..1499
                     /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             1500..2210
                     /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(2227..2251)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2268..2856
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2321..2341)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2739..2750)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3036..3505
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3635..3652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3806..4394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3886..3905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4558..5349,5350..5418))
                     /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     5181..5200
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5419..5523)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5553..6146
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6209..6566
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6625..7041
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6971..6993
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7009..7025
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(7051..7246,1..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 agtcgagtcg actggatcca ccatgggttg
      301 aaccatgggt tgaaccatgg gttgagatcc accatgggtt gaaccatggg ttgaaccatg
      361 ggttgagatc cggtactact gccaccatgg gagacatggg cgatccacca aaaaaaaaac
      421 gtctgatttc cctgtgtgtt ggttgcggca atcaaattca cgaccagtat attctgaggg
      481 tttctccgga tttggagtgg catgcagcat gtttgaaatg tgcggagtgt aatcagtatt
      541 tggacgaaag ctgtacgtgc tttgttaggg atgggaaaac ctactgtaaa agagattata
      601 tcaggttgta cgggatcaaa tgcgccaagt gcagcatagg cttcagcaag aacgacttcg
      661 tgatgcgcgc ccgctctaag gtgtaccaca tcgagtgttt ccgctgtgta gcctgcagcc
      721 gacagctcat cccgggagac gaattcgccc tgcgggagga tgggcttttc tgccgtgcag
      781 accacgatgt ggtggagaga gccagcctgg gagctggaga tcctctcagt cccttgcatc
      841 cagcgcggcc tctgcaaatg gcagccgaac ccatctcggc taggcagcca gctctgcggc
      901 cgcacgtcca caagcagccg gagaagacca cccgagtgcg gactgtgctc aacgagaagc
      961 agctgcacac cttgcggacc tgctatgccg ccaaccctcg gccagatgcg ctcatgaagg
     1021 agcaactagt ggagatgacg ggcctcagtc ccagagtcat ccgagtgtgg tttcaaaaca
     1081 agcggtgcaa ggacaagaaa cgcagcatca tgatgaagca gctccagcag cagcaaccca
     1141 acgacaaaac taatatccag gggatgacag gaactcccat ggtggctgct agtccggaga
     1201 gacatgatgg tggtttacag gctaacccag tagaggtgca aagttaccag ccgccctgga
     1261 aagtactgag tgacttcgcc ttgcaaagcg acatagatca gcctgctttt cagcaactgg
     1321 tcaatttttc agaaggagga ccaggctcta attctactgg cagtgaagta gcatcgatgt
     1381 cctcgcagct cccagataca cccaacagca tggtagccag tcctattgag gcaggaagcg
     1441 gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta
     1501 tggtgtctaa gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag
     1561 gttcggtcaa cggccaccaa ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg
     1621 gaactcaaac catgaggatc aaagtcatcg agggaggacc cctgccattt gcctttgaca
     1681 ttcttgccac gtcgttcatg tatggcagcc gtacttttat caagtacccg aaaggcattc
     1741 ctgatttctt taaacagtcc tttcctgagg gttttacttg ggaaagagtt acgagatacg
     1801 aagatggtgg agtcgtcacc gtcatgcagg acaccagcct tgaggatggc tgtctcgttt
     1861 accacgtcca agtcagaggg gtaaactttc cctccaatgg tcccgtgatg cagaagaaga
     1921 ccaagggttg ggagcctaat acagagatga tgtatccagc agatggtggt ctgaggggat
     1981 acactcatat ggcactgaaa gttgatggtg gtggccatct gtcttgctct ttcgtaacaa
     2041 cttacaggtc aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat gccgttgatc
     2101 accgcctgga aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg
     2161 cagttgccaa gttcgccggg cttggtggtg ggatggacga gctgtacaag taacgctgat
     2221 atctagaccc agctttcttg tacaaagtgg ttcgatagct tatcgataat caacctctgg
     2281 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     2341 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     2401 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     2461 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     2521 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     2581 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     2641 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     2701 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     2761 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     2821 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cgataaaata
     2881 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     2941 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     3001 gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc
     3061 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg
     3121 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     3181 tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     3241 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     3301 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     3361 gcgcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct
     3421 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     3481 actacccgtc agcgggggtc tttcacatgc agcatgtatc aaaattaatt tggttttttt
     3541 tcttaagtat ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga
     3601 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3661 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3721 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3781 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3841 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3901 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3961 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4021 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4081 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4141 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4201 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4261 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4321 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4381 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4441 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4501 ttttgcggcc ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4561 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4621 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4681 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4741 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4801 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4861 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4921 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4981 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5041 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5101 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5161 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5221 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5281 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5341 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5401 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5461 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5521 gcgcacattt ccccgaaaag tgccacctgc ataatgaaag accccacctg taggtttggc
     5581 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa
     5641 aagttcagat caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct
     5701 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg
     5761 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt
     5821 ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc
     5881 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
     5941 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
     6001 cgcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt
     6061 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
     6121 ctacccgtca gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc
     6181 agggaccacc gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg
     6241 attgtctagt gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc
     6301 tctgtatctg gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg
     6361 ggagacgtcc cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc
     6421 gatcgttttg gactctttgg tgcacccccc taataggagg gatatgtggt tctggtagga
     6481 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga
     6541 agccgcgccg cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg
     6601 tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta
     6661 ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga
     6721 cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac
     6781 ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg
     6841 catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc
     6901 cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc
     6961 ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc
     7021 ctcactcctt ctctaggcgc ccccatatgg ccatatgaga tcttatatgg ggcacccccg
     7081 ccccttgtaa acttccctga ccctgacatg acaagagtta ctaacagccc ctctctccaa
     7141 gctcacttac aggctctcta cttagtccag cacgaagtct ggagacctct ggcggcagcc
     7201 taccaagaac aactggaccg accggtggta cctcaccctt accgag
//
