LOCUS       sequence-6944-ex        6963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral construct for miRNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6963)
  AUTHORS   Chen CZ, Li L, Lodish HF, Bartel DP
  TITLE     MicroRNAs modulate hematopoietic lineage differentiation.
  JOURNAL   Science. 2004 Jan 2. 303(5654):83-6.
  PUBMED    14657504
REFERENCE   2  (bases 1 to 6963)
  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: "Science. 
            2004 Jan 2. 303(5654):83-6."
COMMENT     Sequence Label: MDH1-PGK-GFP_2.0
FEATURES             Location/Qualifiers
     source          1..6963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             988..1404
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1334..1356
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1372..1388
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1716..1732
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     promoter        1796..2296
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     2229..2248
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2306..2308,2309..2311,2312..3025)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2351..2372)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2612..2631)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2959..2980
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3168..3258)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(3228..3247)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(3922..3938)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3922..3938)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3935..3957)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3946..3962
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3970..3976,3977..3994,3995..4000))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4015..4036
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4153..4170)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4324..4912)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4404..4423)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5083..5874,5875..5943))
                     /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     5706..5725
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5944..6048)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6116..6134
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6172..6194)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6294..6313
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6507..6529
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6521..6538
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6522..6538
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6741..6760
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6962..6963,1..516)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgccggaatt agatcttata tggggcaccc ccgccccttg
     1441 taaacttccc tgaccctgac atgacaagag ttactaacag cccctctctc caagctcact
     1501 tacaggctct ctacttagtc cagcacgaag tctggagacc tctggcggca gcctaccaag
     1561 aacaactgga ccgaccggtg gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc
     1621 gccgacacca gactaagaac ctagaacctc gctggaaagg accttacaca gtcctgctga
     1681 ccacccccac cgccctcaaa gtagacggca tcgcagcttg gatacacgcc gcccacgtga
     1741 aggctgccga ccccgggggt ggaccatcct ctagactgcc ggatcaattc ctaccgggta
     1801 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcggcccc gctgggcact
     1861 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     1921 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     1981 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     2041 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     2101 agcggccaat agcagctttg gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     2161 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     2221 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     2281 atctccgggc ctttcgacct gcaccatggt gagcaagggc gaggagctgt tcaccggggt
     2341 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2401 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2461 caagctgccc gtgccctggc ccaccctcgt gaccaccttc acctacggcg tgcagtgctt
     2521 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2581 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2641 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2701 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2761 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2821 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2881 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2941 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3001 cggcatggac gagctgtaca agtaatgaat taattaagaa ttgcggccgc gtcgacctgc
     3061 agccaagctt atcgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga
     3121 aagaccccac ctgtaggttt ggcaagaatt cgtttaaacg ggctcgagtg gtctcataca
     3181 gaacttataa gattcccaaa tccaaagaca tttcacgttt atggtgattt cccagaacac
     3241 atagcgacat gcaaatattg ggatccgcta gcttaagtaa cgccattttg caaggcatgg
     3301 aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga
     3361 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     3421 agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc
     3481 agggtgcccc aaggacctga aaatgaccct gtgccttatt tgaactaacc aatcagttcg
     3541 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc
     3601 tcactcggcg cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg tatccaataa
     3661 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
     3721 gtgattgact acccgtcagc gggggtcttt catgggtaac agtttcttga agttggagaa
     3781 caacattctg agggtaggag tcgaatatta agtaatcctg actcaattag ccactgtttt
     3841 gaatccacat actccaatac tcctgaaata gttcattatg gacagcgcag aagagctggg
     3901 gagaattaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3961 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4021 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4081 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4141 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4201 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4261 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4321 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4381 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4441 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4501 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4561 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4621 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4681 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4741 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4801 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4861 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4921 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4981 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5041 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5101 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5161 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5221 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5281 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5341 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5401 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5461 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5521 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5581 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5641 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5701 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5761 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5821 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5881 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5941 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6001 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6061 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     6121 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     6181 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     6241 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     6301 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     6361 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     6421 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     6481 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc
     6541 caagctcgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6601 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6661 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     6721 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6781 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgattagtc caatttgtta
     6841 aagacaggat atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc
     6901 ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag aaaaaggggg
     6961 gaa
//
