LOCUS       sequence-465039-        7399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of mLhx3 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 7399)
  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 7399)
  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-mLhx3-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7399
                     /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             1596..1652
                     /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             1653..2363
                     /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(2380..2404)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2421..3009
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2474..2494)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2892..2903)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3189..3658
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3788..3805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3959..4547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4039..4058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4711..5502,5503..5571))
                     /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     5334..5353
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5572..5676)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5706..6299
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6362..6719
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6778..7194
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7124..7146
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7162..7178
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(7204..7399,1..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 gccaccatgc tgctagaagc agaactcgat tgccaccgag
      421 agaggcccgg tgcccctgga gcttctgccc tctgtacctt cagcaggact ccagagatcc
      481 cgatgtgtgc aggctgtgac cagcacatct tggaccgttt catccttaag gctctggacc
      541 gacattggca cagcaagtgt ctcaagtgca gtgactgcca cgtccctctg gctgagcgct
      601 gcttcagccg cggggagagc gtctactgca aagacgactt ctttaagcgc ttcgggacca
      661 agtgcgccgc atgccagctg ggcatcccgc ccacgcaggt ggtgcgccgc gcccaggact
      721 tcgtgtacca cctgcattgc ttcgcctgcg tggtctgcaa gcggcagctg gccacgggcg
      781 acgagttcta cctcatggaa gacagccggc tggtgtgcaa ggcggactac gaaacagcca
      841 agcagcgaga agccgaggcc acagccaagc ggccgcgcac caccatcacc gccaagcagc
      901 tggagacgct gaagagcgcc tacaacactt cgcccaagcc ggcgcgccac gtgcgcgagc
      961 agctctcctc cgagactggc ctggacatgc gagtggtgca ggtgtggttc cagaatcgcc
     1021 gggctaagga aaagagactg aagaaagacg ctggccggca gcgctgggga cagtatttcc
     1081 gcaatatgaa gcgctcccgc ggcagttcca agtccgacaa ggacagcatc caggagggac
     1141 aagacagcga cgccgaagtc tccttcactg atgagccgtc catggctgac atggggcctg
     1201 ctaatggcct gtacagcagc ctgggagagc ctgcccctgc gttgggccgg cccgtaggag
     1261 gcctgggcag ctttaccctg gatcacggag gcttgacggg tccagagcag taccgagagc
     1321 tacgcccagg cagcccctat ggcatccccc catctcctgc agccccccag agccttcctg
     1381 gcccccagcc tctcctctcc agcctggtat acccagacac caacttgagc cttgtccctt
     1441 cagggccccc aggtggaccc ccacccatga gggtgctggc tggaaatggg cccagctccg
     1501 acctgtccac agagagcagt tctggctacc cagactttcc tgctagccct gcttcctggc
     1561 tggatgaagt agaccatgct cagttcggaa gcggagctac taacttcagc ctgctgaagc
     1621 aggctggaga cgtggaggag aaccctggac ctatggtgtc taagggcgaa gagctgatca
     1681 aggaaaatat gcgtatgaag gtggtcatgg aaggttcggt caacggccac caattcaaat
     1741 gcacaggtga aggagaaggc aatccgtaca tgggaactca aaccatgagg atcaaagtca
     1801 tcgagggagg acccctgcca tttgcctttg acattcttgc cacgtcgttc atgtatggca
     1861 gccgtacttt tatcaagtac ccgaaaggca ttcctgattt ctttaaacag tcctttcctg
     1921 agggttttac ttgggaaaga gttacgagat acgaagatgg tggagtcgtc accgtcatgc
     1981 aggacaccag ccttgaggat ggctgtctcg tttaccacgt ccaagtcaga ggggtaaact
     2041 ttccctccaa tggtcccgtg atgcagaaga agaccaaggg ttgggagcct aatacagaga
     2101 tgatgtatcc agcagatggt ggtctgaggg gatacactca tatggcactg aaagttgatg
     2161 gtggtggcca tctgtcttgc tctttcgtaa caacttacag gtcaaaaaag accgtcggga
     2221 acatcaagat gcccggtatc catgccgttg atcaccgcct ggaaaggtta gaggaaagtg
     2281 acaatgaaat gttcgtagta caacgcgaac acgcagttgc caagttcgcc gggcttggtg
     2341 gtgggatgga cgagctgtac aagtaacgct gatatctaga cccagctttc ttgtacaaag
     2401 tggttcgata gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     2461 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2521 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2581 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2641 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2701 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2761 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     2821 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     2881 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     2941 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3001 cctccccgca tcgataccgt cgacgataaa ataaaagatt ttatttagtc tccagaaaaa
     3061 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg
     3121 caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga
     3181 tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
     3241 agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
     3301 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca
     3361 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc
     3421 cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg
     3481 agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga ttgactgagt
     3541 cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg
     3601 ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcaca
     3661 tgcagcatgt atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat
     3721 agttgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3781 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3841 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3901 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3961 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4021 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4081 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4141 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4201 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4261 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4321 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4381 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4441 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4501 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4561 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4621 tgagattatc aaaaaggatc ttcacctaga tccttttgcg gccggccgca aatcaatcta
     4681 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4741 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4801 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4861 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4921 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4981 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5041 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5101 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5161 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5221 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5281 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5341 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5401 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5461 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5521 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5581 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5641 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5701 tgcataatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc
     5761 aaggcatgga aaaatacata actgagaata gaaaagttca gatcaaggtc aggaacagat
     5821 ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     5881 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
     5941 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
     6001 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
     6061 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
     6121 gctcaataaa agagcccaca acccctcact cggcgcgcca gtcctccgat tgactgagtc
     6181 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
     6241 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
     6301 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
     6361 taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
     6421 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     6481 ctgacgagtt cggaacaccc ggccgcaacc ctgggagacg tcccagggac ttcgggggcc
     6541 gtttttgtgg cccgacctga gtccaaaaat cccgatcgtt ttggactctt tggtgcaccc
     6601 ccctaatagg agggatatgt ggttctggta ggagacgaga acctaaaaca gttcccgcct
     6661 ccgtctgaat ttttgctttc ggtttgggac cgaagccgcg ccgcgcgtct tgtctgctgc
     6721 agcatcgttc tgtgttgtct ctgtctgact gtgtttctgt atttgtctga aaattagggc
     6781 cagactgtta ccactccctt aagtttgacc ttaggtcact ggaaagatgt cgagcggatc
     6841 gctcacaacc agtcggtaga tgtcaagaag agacgttggg ttaccttctg ctctgcagaa
     6901 tggccaacct ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc
     6961 caggttaaga tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac
     7021 atcgtgacct gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac
     7081 cctaagcctc cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt
     7141 tcgaccccgc ctcgatcctc cctttatcca gccctcactc cttctctagg cgcccccata
     7201 tggccatatg agatcttata tggggcaccc ccgccccttg taaacttccc tgaccctgac
     7261 atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc
     7321 cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg
     7381 gtacctcacc cttaccgag
//
