LOCUS       sequence-254541-        3583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP entry clones.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3583)
  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 3583)
  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: S8_attL1-N-term-EGFP-attL2
FEATURES             Location/Qualifiers
     source          1..3583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..52)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     152..171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     365..387
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     379..396
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     380..396
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    446..545
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(548..550,551..553,554..1267)
                     /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(593..614)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(854..873)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1201..1222
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1274..1373)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1408..1424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1408..1424)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1421..1443)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1432..1448
                     /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(1456..1462,1463..1480,1481..1486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1501..1522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1639..1656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1810..2398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1890..1909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2569..3384)
                     /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             complement(2569..3384)
                     /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     3292..3311
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3385..3489)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3557..3575
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
       61 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      121 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      181 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
      241 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
      301 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
      361 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgacgcgtat tgggatcagt
      421 gaattgacgc gtattgggat ggaaccaaat aatgatttta ttttgactga tagtgacctg
      481 ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt acaaaaaagc
      541 aggctccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
      601 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
      661 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
      721 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
      781 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
      841 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
      901 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
      961 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1021 gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1081 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1141 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1201 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1261 caagtagtaa tgaacccagc tttcttgtac aaagttggca ttataagaaa gcattgctta
     1321 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgatcccaa
     1381 tggcgcgccg agcttggctc gagcatggtc atagctgttt cctgtgtgaa attgttatcc
     1441 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     1501 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1561 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1621 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1681 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1741 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1801 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1861 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1921 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1981 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2041 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2101 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2161 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2221 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2281 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2341 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2401 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2461 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2521 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt agaaaaactc
     2581 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     2641 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     2701 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     2761 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     2821 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     2881 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     2941 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     3001 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     3061 ctggaatgct gttttcccag ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     3121 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     3181 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     3241 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     3301 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     3361 cgtttcccgt tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca
     3421 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3481 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     3541 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgt
//
