LOCUS       sequence-256205-        3571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP entry clone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3571)
  AUTHORS   Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D,
            Eggan K, Barrett LE
  TITLE     A multiplexed gRNA piggyBac transposon system facilitates efficient 
            induction of CRISPRi and CRISPRa in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2020 Jan 20;10(1):635. doi: 10.1038/s41598-020-57500-1.
  PUBMED    31959800
REFERENCE   2  (bases 1 to 3571)
  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: "Sci Rep."; 
            date: "2020-01-20"; volume: "10(1):635. doi"; pages: " 
            10.1038/s41598-020-57500-1"
COMMENT     Sequence Label: S9_attL1-EGFP-C-term-attL2
FEATURES             Location/Qualifiers
     source          1..3571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    52..151
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      172..181
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(178..180,181..183,184..894)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(223..244)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(484..503)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     831..852
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(896..995)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1017..1034)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1017..1033)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1026..1048)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1242..1261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1361..1383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1421..1439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1507..1611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1612..2427
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1612..2427
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1685..1704)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2598..3186
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3087..3106
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3340..3357
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3474..3495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3510..3515,3516..3533,3534..3540)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3548..3564
                     /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)."
     primer_bind     join(3553..3571,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gctcgagcca agctcggcgc gccattggga tcaaataatg
       61 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca atgctttttt
      121 ataatgccaa ctttgtacaa aaaagcaggc taagctggct agttaagctt cgccaccatg
      181 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      241 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      301 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      361 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      421 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      481 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      541 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      601 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      661 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      721 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      781 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      841 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcaccca
      901 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
      961 caggtcacta tcagtcaaaa taaaatcatt atttgatccc aatggcgcgt caattcactg
     1021 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1081 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1141 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     1201 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     1261 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     1321 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1381 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     1441 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     1501 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1561 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagccat
     1621 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     1681 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     1741 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     1801 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     1861 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccctggg
     1921 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     1981 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     2041 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     2101 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     2161 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     2221 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     2281 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     2341 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     2401 catttgatgc tcgatgagtt tttctaactg tcagaccaag tttactcata tatactttag
     2461 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2521 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2581 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2641 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2701 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     2761 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2821 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2881 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2941 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3001 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3061 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3121 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3181 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3241 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3301 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3361 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3421 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     3481 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     3541 tgtggaattg tgagcggata acaatttcac a
//
