LOCUS       sequence-442485-        6475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express TagBFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6475)
  TITLE     Single-base tiled screen unveils design principles of PspCas13b for 
            potent and off-target-free RNA silencing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6475)
  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: 
            "Unpublished"
COMMENT     Sequence Label: MSCV-IRES-tagBFP
FEATURES             Location/Qualifiers
     source          1..6475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..48
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     176..195
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             395..911
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    975..1316
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1383..1799
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1729..1751
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1767..1783
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1844..2393
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1988..2005)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2215..2234
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2402..3103
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     LTR             3264..3778
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3940..3956
                     /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(3964..3970,3971..3988,3989..3994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4009..4030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4147..4164)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4318..4906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4398..4417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5077..5868,5869..5937))
                     /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     5700..5719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5938..6042)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6110..6128
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6166..6188)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6288..6307
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga
       61 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
      121 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
      181 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
      241 gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc
      301 tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat
      361 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
      421 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aatacataac tgagaataga
      481 gaagttcaga tcaaggttag gaacagagag acagcagaat atgggccaaa caggatatct
      541 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc
      601 cgccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa
      661 tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct
      721 tctgctcccc gagctcaata aaagagccca caacccctca ctcggcgcgc cagtcctccg
      781 atagactgcg tcgcccgggt acccgtattc ccaataaagc ctcttgctgt ttgcatccga
      841 atcgtggact cgctgatcct tgggagggtc tcctcagatt gattgactgc ccacctcggg
      901 ggtctttcat ttggaggttc caccgagatt tggagacccc tgcccaggga ccaccgaccc
      961 ccccgccggg aggtaagctg gccagcggtc gtttcgtgtc tgtctctgtc tttgtgcgtg
     1021 tttgtgccgg catctaatgt ttgcgcctgc gtctgtacta gttagctaac tagctctgta
     1081 tctggcggac ccgtggtgga actgacgagt tctgaacacc cggccgcaac cctgggagac
     1141 gtcccaggga ctttgggggc cgtttttgtg gcccgacctg aggaagggag tcgatgtgga
     1201 atccgacccc gtcaggatat gtggttctgg taggagacga gaacctaaaa cagttcccgc
     1261 ctccgtctga atttttgctt tcggtttgga accgaagccg cgcgtcttgt ctgctgcagc
     1321 gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt
     1381 agggccagac tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc
     1441 ggatcgctca caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg
     1501 cagaatggcc aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca
     1561 tcacccaggt taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc
     1621 cctacatcgt gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg
     1681 tacaccctaa gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc
     1741 ctcgttcgac cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccg
     1801 gaattcgtta acctcgagcg ggatcaattc cgcccccccc ctaacgttac tggccgaagc
     1861 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     1921 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     1981 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     2041 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
     2101 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
     2161 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
     2221 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
     2281 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
     2341 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgata ataccatggc
     2401 catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     2461 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     2521 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     2581 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     2641 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aggacggggg
     2701 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     2761 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     2821 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     2881 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     2941 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     3001 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     3061 atactgcgac ctccctagca aactggggca caagcttaat tgagcggccg cgactctaga
     3121 gtcgacctgc aggcatgcaa gcttcaggta gccggctaac gttaacaacc ggtacctcta
     3181 gaactatagc tagcatgcgc aaatttaaag cgctgatatc gataaaataa aagattttat
     3241 ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt
     3301 aagtaacgcc attttgcaag gcatggaaaa tacataactg agaatagaga agttcagatc
     3361 aaggttagga acagagagac agcagaatat gggccaaaca ggatatctgt ggtaagcagt
     3421 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtcccg ccctcagcag
     3481 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
     3541 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
     3601 gctcaataaa agagcccaca acccctcact cggcgcgcca gtcctccgat agactgcgtc
     3661 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
     3721 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcatgg
     3781 gtaacagttt cttgaagttg gagaacaaca ttctgagggt aggagtcgaa tattaagtaa
     3841 tcctgactca attagccact gttttgaatc cacatactcc aatactcctg aaatagttca
     3901 ttatggacag cgcagaaaga gctggggaga attgtgaaat tgttatccgc tcacaattcc
     3961 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4021 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4081 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4141 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4201 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4261 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4321 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4381 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4441 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4501 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4561 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4621 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4681 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4741 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4801 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4861 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4921 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4981 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5041 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5101 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5161 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5221 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5281 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5341 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5401 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5461 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5521 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5581 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5641 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5701 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5761 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5821 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5881 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5941 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6001 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6061 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     6121 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     6181 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     6241 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
     6301 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     6361 ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     6421 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcga
//
