LOCUS       sequence-176747-        6116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP reporter cell line with eight bulged miR-19 sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6116)
  AUTHORS   Golden RJ, Chen B, Li T, Braun J, Manjunath H, Chen X, Wu J, Schmid 
            V, Chang TC, Kopp F, Ramirez-Martinez A, Tagliabracci VS, Chen ZJ, 
            Xie Y, Mendell JT
  TITLE     An Argonaute phosphorylation cycle promotes microRNA-mediated 
            silencing.
  JOURNAL   Nature. 2017 Feb 9;542(7640):197-202. doi: 10.1038/nature21025. Epub
            2017 Jan 23.
  PUBMED    28114302
REFERENCE   2  (bases 1 to 6116)
  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"; doi: 
            "10.1038/nature21025"; journalName: "Nature"; date: "2017-02-9- 9"; 
            volume: "542"; issue: "7640"; pages: "197-202"
COMMENT     Sequence Label: MSCV-EGFP-Bulged-miR-19
FEATURES             Location/Qualifiers
     source          1..6116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             join(1439..1441,1442..1444,1445..2158)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1439..1441,1442..1444,1445..2155)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1484..1505)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1745..1764)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2092..2113
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             2478..2992
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(3161..3177)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3161..3177)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3174..3196)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3185..3201
                     /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(3209..3215,3216..3233,3234..3239))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3254..3275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3392..3409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3563..4151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3643..3662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4322..5113,5114..5182))
                     /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     4945..4964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5183..5287)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5355..5373
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5411..5433)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5533..5552
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5746..5768
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5896..5915
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6115..6116,1..515)
                     /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 gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ggttctgaga gatctaccat
     1441 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1501 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1561 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1621 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1681 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1741 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1801 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1861 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1921 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1981 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2041 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2101 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaact
     2161 cgagctcaga acgaattcct ggttaacgac gggtccctca gttttgccct atttgcacaa
     2221 atttcagttt tgccctattt gcacagggtc cctcagtttt gccctatttg cacaaatttc
     2281 agttttgccc tatttgcaca gggtccctca gttttgccct atttgcacaa atttcagttt
     2341 tgccctattt gcacagggtc cctcagtttt gccctatttg cacaaatttc agttttgccc
     2401 tatttgcaca gggtcccgac gtttaaacga catcgataaa ataaaagatt ttatttagtc
     2461 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa
     2521 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt
     2581 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     2641 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta
     2701 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     2761 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     2821 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg
     2881 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     2941 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca
     3001 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga
     3061 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg
     3121 acagcgcaga agagctgggg agaattaatt cgtaatcatg gtcatagctg tttcctgtgt
     3181 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3241 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3301 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3361 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3421 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3481 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3541 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3601 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3661 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3721 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3781 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3841 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3901 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3961 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4021 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4081 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4141 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4201 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4261 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4321 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4381 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4441 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4501 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4561 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4621 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4681 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4741 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4801 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4861 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4921 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4981 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5041 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5101 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5161 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5221 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5281 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5341 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     5401 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     5461 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     5521 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     5581 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
     5641 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     5701 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     5761 gtaaaacgac ggcgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5821 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     5881 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     5941 ggtgatgccg gccacgatgc gtccggcgta gaggcgatta gtccaatttg ttaaagacag
     6001 gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga
     6061 gtacgagcca tagataaaat aaaagatttt atttagtctc cagaaaaagg ggggaa
//
