LOCUS       sequence-463007-        7329 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 7329)
  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 7329)
  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-uORFwx1-mLhx3-P2A-mRuby2
FEATURES             Location/Qualifiers
     source          1..7329
                     /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             1526..1582
                     /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             1583..2293
                     /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(2310..2334)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2351..2939
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2404..2424)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2822..2833)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3119..3588
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3718..3735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3889..4477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3969..3988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4641..5432,5433..5501))
                     /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     5264..5283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5502..5606)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5636..6229
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6292..6649
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6708..7124
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7054..7076
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7092..7108
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    join(7134..7329,1..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 atcccgaatg ggttgagatc
      301 cggtactact gccaccatgc tgctagaagc agaactcgat tgccaccgag agaggcccgg
      361 tgcccctgga gcttctgccc tctgtacctt cagcaggact ccagagatcc cgatgtgtgc
      421 aggctgtgac cagcacatct tggaccgttt catccttaag gctctggacc gacattggca
      481 cagcaagtgt ctcaagtgca gtgactgcca cgtccctctg gctgagcgct gcttcagccg
      541 cggggagagc gtctactgca aagacgactt ctttaagcgc ttcgggacca agtgcgccgc
      601 atgccagctg ggcatcccgc ccacgcaggt ggtgcgccgc gcccaggact tcgtgtacca
      661 cctgcattgc ttcgcctgcg tggtctgcaa gcggcagctg gccacgggcg acgagttcta
      721 cctcatggaa gacagccggc tggtgtgcaa ggcggactac gaaacagcca agcagcgaga
      781 agccgaggcc acagccaagc ggccgcgcac caccatcacc gccaagcagc tggagacgct
      841 gaagagcgcc tacaacactt cgcccaagcc ggcgcgccac gtgcgcgagc agctctcctc
      901 cgagactggc ctggacatgc gagtggtgca ggtgtggttc cagaatcgcc gggctaagga
      961 aaagagactg aagaaagacg ctggccggca gcgctgggga cagtatttcc gcaatatgaa
     1021 gcgctcccgc ggcagttcca agtccgacaa ggacagcatc caggagggac aagacagcga
     1081 cgccgaagtc tccttcactg atgagccgtc catggctgac atggggcctg ctaatggcct
     1141 gtacagcagc ctgggagagc ctgcccctgc gttgggccgg cccgtaggag gcctgggcag
     1201 ctttaccctg gatcacggag gcttgacggg tccagagcag taccgagagc tacgcccagg
     1261 cagcccctat ggcatccccc catctcctgc agccccccag agccttcctg gcccccagcc
     1321 tctcctctcc agcctggtat acccagacac caacttgagc cttgtccctt cagggccccc
     1381 aggtggaccc ccacccatga gggtgctggc tggaaatggg cccagctccg acctgtccac
     1441 agagagcagt tctggctacc cagactttcc tgctagccct gcttcctggc tggatgaagt
     1501 agaccatgct cagttcggaa gcggagctac taacttcagc ctgctgaagc aggctggaga
     1561 cgtggaggag aaccctggac ctatggtgtc taagggcgaa gagctgatca aggaaaatat
     1621 gcgtatgaag gtggtcatgg aaggttcggt caacggccac caattcaaat gcacaggtga
     1681 aggagaaggc aatccgtaca tgggaactca aaccatgagg atcaaagtca tcgagggagg
     1741 acccctgcca tttgcctttg acattcttgc cacgtcgttc atgtatggca gccgtacttt
     1801 tatcaagtac ccgaaaggca ttcctgattt ctttaaacag tcctttcctg agggttttac
     1861 ttgggaaaga gttacgagat acgaagatgg tggagtcgtc accgtcatgc aggacaccag
     1921 ccttgaggat ggctgtctcg tttaccacgt ccaagtcaga ggggtaaact ttccctccaa
     1981 tggtcccgtg atgcagaaga agaccaaggg ttgggagcct aatacagaga tgatgtatcc
     2041 agcagatggt ggtctgaggg gatacactca tatggcactg aaagttgatg gtggtggcca
     2101 tctgtcttgc tctttcgtaa caacttacag gtcaaaaaag accgtcggga acatcaagat
     2161 gcccggtatc catgccgttg atcaccgcct ggaaaggtta gaggaaagtg acaatgaaat
     2221 gttcgtagta caacgcgaac acgcagttgc caagttcgcc gggcttggtg gtgggatgga
     2281 cgagctgtac aagtaacgct gatatctaga cccagctttc ttgtacaaag tggttcgata
     2341 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     2401 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     2461 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     2521 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     2581 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     2641 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     2701 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     2761 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     2821 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     2881 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     2941 tcgataccgt cgacgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     3001 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     3061 aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct
     3121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     3181 acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     3241 ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag
     3301 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     3361 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     3421 aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta
     3481 cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
     3541 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcaca tgcagcatgt
     3601 atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat agttgcatta
     3661 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3721 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3781 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3841 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3901 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3961 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4021 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4081 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4141 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4201 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4261 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4321 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4381 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4441 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4501 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4561 aaaaaggatc ttcacctaga tccttttgcg gccggccgca aatcaatcta aagtatatat
     4621 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4681 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4741 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4801 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4861 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4921 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4981 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5041 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5101 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5161 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5221 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5281 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5341 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5401 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5461 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5521 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5581 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgcataatga
     5641 aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga
     5701 aaaatacata actgagaata gaaaagttca gatcaaggtc aggaacagat ggaacagctg
     5761 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     5821 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     5881 gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga
     5941 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     6001 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     6061 agagcccaca acccctcact cggcgcgcca gtcctccgat tgactgagtc gcccgggtac
     6121 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     6181 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt
     6241 ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc
     6301 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta tgcgcctgcg
     6361 tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
     6421 cggaacaccc ggccgcaacc ctgggagacg tcccagggac ttcgggggcc gtttttgtgg
     6481 cccgacctga gtccaaaaat cccgatcgtt ttggactctt tggtgcaccc ccctaatagg
     6541 agggatatgt ggttctggta ggagacgaga acctaaaaca gttcccgcct ccgtctgaat
     6601 ttttgctttc ggtttgggac cgaagccgcg ccgcgcgtct tgtctgctgc agcatcgttc
     6661 tgtgttgtct ctgtctgact gtgtttctgt atttgtctga aaattagggc cagactgtta
     6721 ccactccctt aagtttgacc ttaggtcact ggaaagatgt cgagcggatc gctcacaacc
     6781 agtcggtaga tgtcaagaag agacgttggg ttaccttctg ctctgcagaa tggccaacct
     6841 ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc caggttaaga
     6901 tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac atcgtgacct
     6961 gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac cctaagcctc
     7021 cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt tcgaccccgc
     7081 ctcgatcctc cctttatcca gccctcactc cttctctagg cgcccccata tggccatatg
     7141 agatcttata tggggcaccc ccgccccttg taaacttccc tgaccctgac atgacaagag
     7201 ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc cagcacgaag
     7261 tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg gtacctcacc
     7321 cttaccgag
//
