LOCUS       sequence-66626-e        6466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Target EGFP gene that is stably integrated into HEK293 genome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6466)
  AUTHORS   Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, 
            Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, 
            Weissman JS, Qi LS
  TITLE     CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in 
            Eukaryotes.
  JOURNAL   Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 
            10.1016/j.cell.2013.06.044.
  PUBMED    23849981
REFERENCE   2  (bases 1 to 6466)
  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. 2013 
            Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044."
COMMENT     Sequence Label: pMLS-SV40-EGFP
FEATURES             Location/Qualifiers
     source          1..6466
                     /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"
     ncRNA           1443..1536
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1550..1662
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(1695..1715)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1700..2029
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1880..2015
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(2036..2038,2039..2041,2042..2755)
                     /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(2036..2038,2039..2041,2042..2752)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2081..2102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2342..2361)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2689..2710
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             2828..3342
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(3511..3527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3511..3527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3524..3546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3535..3551
                     /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(3559..3565,3566..3583,3584..3589))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3604..3625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3742..3759)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3913..4501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3993..4012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4672..5463,5464..5532))
                     /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     5295..5314
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5533..5637)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5705..5723
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5761..5783)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5883..5902
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6096..6118
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6246..6265
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6465..6466,1..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 ctagggataa cagggtaatt
     1441 gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc
     1501 tgggattact tcttcaggtt aacccaacag aaggctcgag caaccagaat tcaaggggct
     1561 actttaggag caattatctt gtttactaaa actgaatacc ttgctatctc tttgatacat
     1621 ttttacaaag ctgaattaaa atggtataaa ttaaatcact tttttcaatt cgccagcaca
     1681 gtggtcgacc ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     1741 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     1801 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     1861 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     1921 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     1981 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcatggt
     2041 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2101 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2161 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2221 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2281 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2341 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2401 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2461 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2521 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2581 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2641 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2701 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaatgaat
     2761 taattaagaa ttatcaagct tatcgataaa ataaaagatt ttatttagtc tccagaaaaa
     2821 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg
     2881 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag
     2941 agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     3001 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc
     3061 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca
     3121 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc
     3181 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt
     3241 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt
     3301 ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca gtttcttgaa
     3361 gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga ctcaattagc
     3421 cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg acagcgcaga
     3481 agagctgggg agaattaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3541 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3601 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3661 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3721 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3781 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3841 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3901 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3961 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4021 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4081 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4141 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4201 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4261 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4321 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     4381 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4441 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4501 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4561 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4621 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4681 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4741 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4801 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4861 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4921 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4981 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5041 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5101 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5161 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5221 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5281 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5341 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5401 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5461 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5521 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5581 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5641 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     5701 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     5761 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     5821 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     5881 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     5941 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
     6001 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     6061 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     6121 ggcgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6181 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6241 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6301 gccacgatgc gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg
     6361 gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca
     6421 tagataaaat aaaagatttt atttagtctc cagaaaaagg ggggaa
//
