LOCUS       sequence-205524-        6037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP, puromycin and gRNA scaffold driven by EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6037)
  AUTHORS   Pflueger C, Tan D, Swain T, Nguyen TV, Pflueger J, Nefzger C, Polo 
            JM, Ford E, Lister R
  TITLE     A modular dCas9-SunTag DNMT3A epigenome editing system overcomes 
            pervasive off-target activity of direct fusion dCas9-DNMT3A 
            constructs.
  JOURNAL   Genome Res. 2018 Jun 15. pii: gr.233049.117. doi: 
            10.1101/gr.233049.117.
  PUBMED    29907613
REFERENCE   2  (bases 1 to 6037)
  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: "Genome Res.
            2018 Jun 15. pii: gr.233049.117. doi: 10.1101/gr.233049.117."
COMMENT     Sequence Label: LLP109 pEF1a-gRNA-GFP
FEATURES             Location/Qualifiers
     source          1..6037
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        281..1459
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          512..1450
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1407..1427
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1466..1475
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1472..1474,1475..1477,1478..2188)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1472..1474,1475..1477,1478..2188)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1517..1538)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1778..1797)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2125..2146
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2198..2251
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2252..2848
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2745..2765
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     intron          2887..3116
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3099..3118
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     polyA_signal    3295..3416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3332..3351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3551..3571
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3722..3741
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3880..3896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3880..3896)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3893..3915)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3904..3920
                     /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(3928..3934,3935..3952,3953..3958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3973..3994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4111..4128)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4282..4870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4362..4381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5041..5832,5833..5901))
                     /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     5664..5683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5902..6006)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg tgcaaagatg gataaagttt taaacagaga ggaatctttg
      241 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
      301 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      361 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
      421 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
      481 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
      541 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
      601 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
      661 tcaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
      721 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
      781 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
      841 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
      901 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
      961 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     1021 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     1081 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     1141 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1201 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1261 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1321 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1381 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     1441 tccatttcag gtgtcgtgac gtacggccac catggtgagc aagggcgagg agctgttcac
     1501 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1561 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1621 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1681 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1741 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1801 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1861 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1921 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1981 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2041 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2101 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2161 cactctcggc atggacgagc tgtacaaggg cagtggagag ggcagaggaa gtctgctaac
     2221 atgcggtgac gtcgaggaga atcctggccc aaccgagtac aagcccacgg tgcgcctcgc
     2281 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     2341 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
     2401 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     2461 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     2521 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     2581 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtgtc
     2641 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     2701 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     2761 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     2821 gtgcatgacc cgcaagcccg gtgcctgatg gaattaattc gctgtctgcg agggccagct
     2881 gttggggtga gtactccctc tcaaaagcgg gcatgacttc tgcgctaaga ttgtcagttt
     2941 ccaaaaacga ggaggatttg atattcacct ggcccgcggt gatgcctttg agggtggccg
     3001 cgtccatctg gtcagaaaag acaatctttt tgttgtcaag cttgaggtgt ggcaggcttg
     3061 agatctggcc atacacttga gtgacaatga catccacttt gcctttctct ccacaggtgt
     3121 ccactcccag gtccaactgc aggtcgattt aaatgggaat tccacagcgg ccgcatgtct
     3181 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3241 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3301 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3361 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaatgc
     3421 gtcgagtgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3481 ttgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt
     3541 cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     3601 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     3661 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     3721 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     3781 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     3841 tttaccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3901 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3961 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4021 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4081 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4141 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4201 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4261 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4321 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4381 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4441 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4501 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4561 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4621 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4681 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4741 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4801 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4861 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4921 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4981 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5041 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5101 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5161 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5221 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5281 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5341 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5401 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5461 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5521 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5581 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5641 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5701 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5761 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5821 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5881 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5941 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6001 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
