LOCUS       sequence-325845-        5663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Introduce constitutive (EF1a promoter) GFP cDNA into (ES) cells by 
            recombination-mediated cassette exchange.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5663)
  TITLE     Karreth lab plasmids
REFERENCE   2  (bases 1 to 5663)
  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"
COMMENT     Sequence Label: cEF1a-GFP
FEATURES             Location/Qualifiers
     source          1..5663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..547
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(83..106)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     480..499
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    593..640
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_feature    join(673..738,739..776)
                     /label=Ad SA
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
     polyA_signal    945..1079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1199..1333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1349..1560
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1562..1571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1568..1570,1571..1573,1574..2287)
                     /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(1568..1570,1571..1573,1574..2284)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1613..1634)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1874..1893)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2221..2242
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          2330..2902
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     rep_origin      complement(3230..3818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3310..3329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3989..4780,4781..4849))
                     /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     4612..4631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4850..4954)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4980..5435)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5117..5138)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5329..5348
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5562..5584
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5576..5593
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5577..5593
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5600..5619
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5600..5618
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     join(5651..5663,1..4)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5651..5663,1..4)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 gatcccccgg gctgcaggaa ttaattcgtc gacctcgaaa ttctaccggg taggggaggc
       61 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
      121 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
      181 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
      241 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
      301 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
      361 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
      421 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
      481 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
      541 cctttcgacc tgcatccatc tagatctcga tcgagcagct gaagcttatg gagaagttac
      601 tattccgaag ttcctattct ctagaaagta taggaacttc gatccccctc gatcccccgg
      661 gctgcagatc tgtagggcgc agtagtccag ggtttccttg atgatgtcat acttatcctg
      721 tccctttttt ttccacagct cgcggttgag gacaaactct tcgcggtctt tccagtgggg
      781 atcgacggta tcgataagct tgcagatctg cgactctaga ggatctgcga ctctagagga
      841 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
      901 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
      961 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1021 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct
     1081 gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1141 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1201 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1261 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1321 tcatgtctgg atcgagttta ccatgcatgg gcagagcgca catcgcccac agtccccgag
     1381 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
     1441 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     1501 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     1561 tgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1621 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1681 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1741 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     1801 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1861 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1921 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1981 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2041 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2101 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2161 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2221 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2281 caagtaactc gagttcatga attcgcagtc gagcgttgag aacttcaggg tgagtttggg
     2341 gacccttgat tgttctttct ttttcgctat tgtaaaattc atgttatatg gagggggcaa
     2401 agttttcagg gtgttgttta gaatgggaag atgtcccttg tatcaccatg gaccctcatg
     2461 ataattttgt ttctttcact ttctactctg ttgacaacca ttgtctcctc ttattttctt
     2521 ttcattttct gtaacttttt cgttaaactt tagcttgcat ttgtaacgaa tttttaaatt
     2581 cacttttgtt tatttgtcag attgtaagta ctttctctaa tcactttttt ttcaaggcaa
     2641 tcagggtata ttatattgta cttcagcaca gttttagaga acaattgtta taattaaatg
     2701 ataaggtaga atatttctgc atataaattc tggctggcgt ggaaatattc ttattggtag
     2761 aaacaactac accctggtca tcatcctgcc tttctcttta tggttacaat gatatacact
     2821 gtttgagatg aggataaaat actctgagtc caaaccgggc ccctctgcta accatgttca
     2881 tgccttcttc tctttcctac agctcctggg caacgtgctg gttgttgtgc tgtctcatca
     2941 ttttggcaaa ggattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
     3001 ggccaatgcc ctggctcaca aataccactg agatcgtttt ccctctgcca aaaattatgg
     3061 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     3121 tgcaatagtg tgttggaatt tctcgatcgc tagcatccta ggcgcgccac atgtgagcaa
     3181 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3241 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3301 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3361 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3421 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3481 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3541 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3601 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3661 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3721 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3781 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3841 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3901 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3961 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4021 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4081 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4141 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4201 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4261 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4321 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4381 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4441 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4501 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4561 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4621 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4681 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4741 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4801 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4861 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4921 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     4981 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5041 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5101 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5161 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5221 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5281 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5341 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5401 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     5461 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     5521 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     5581 aacgacggcc agtgaattgt aatacgactc actatagggc gaattggagc tccaccgcgg
     5641 tggcggccgc tctagaacta gtg
//
