LOCUS       sequence-12870-e        7496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7496)
  AUTHORS   Mosimann C, Kaufman CK, Li P, Pugach EK, Tamplin OJ, Zon LI
  TITLE     Ubiquitous transgene expression and Cre-based recombination driven 
            by the ubiquitin promoter in zebrafish.
  JOURNAL   Development. 2011 Jan . 138(1):169-77.
  PUBMED    21138979
REFERENCE   2  (bases 1 to 7496)
  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: 
            "Development. 2011 Jan . 138(1):169-77."
COMMENT     Sequence Label: pENTR5'_ubi:loxP-EGFP-loxP
FEATURES             Location/Qualifiers
     source          1..7496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    593..688
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(4210..4243)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4250..4259
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4256..4258,4259..4261,4262..4975)
                     /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(4256..4258,4259..4261,4262..4972)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4301..4322)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4562..4581)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4909..4930
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5098..5219
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5135..5154)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5189..5208
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5363..5384)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5473..5506)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5538..5661
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5737..5756)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5738..5756)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5761..5777)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5761..5777)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5879..5898)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5890..6699
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5957..5976)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6846..7434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7335..7354
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccga gttaacgcta ccatggagct ccaaataatg
      601 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgctttttt
      661 ataatgccaa ctttgtatag aaaagttggc tccgaattcg cccttaaact cgagaccagc
      721 aaagttctag aatttgtcga aacatttatg ttatatattt cctgaaaaaa attctgagta
      781 agttcttaag tgttattgcc agcaacataa acaacagacg gcaaaatgaa taaatgataa
      841 caaagcagta ggcttaaata aacctaattt ttataggctg ttctctacaa ccctcaaaca
      901 gtgattagtt ttgtacttat aaacttgccc tttcattcat atttcaagaa aattggttca
      961 gaagatctgg atattctagc agttgttcaa gctcatggag ggatcagtga cctgattcca
     1021 caatgactag gcctaatcca gaaattagat gactgtcaac ataaaaaggc acagcactca
     1081 ctagctgccc tatatatttt attatatttt acatatatta ttttatttat ttagctctga
     1141 gtgctgtact ttctggttaa agaaaactgc ttacaacagc taacctgtac tacctcaggc
     1201 tcagggaatt tggaacaggt ttgtctggtt tgtttcttta accatgcatg cttgttttca
     1261 actatggcaa cacagtcaca tgggacatta cagaaatgat ttgtcgatga catgcgactt
     1321 ttctttaata aagcgcaaag atcccaaaaa gcaaactttt aacaaaaatc atataattat
     1381 attttcaatc cagctttgta gcaactttgt gctgctgttc actcagcaac agatagtcag
     1441 tataaggtca gtgtgtctca aagcagtgcc atctgtttca cacattgcgt tctatatata
     1501 agtgtgctgg ttgacacgac actgtataag gcctaggcta aaacacaaac aatgtagaat
     1561 gacactgtgt tttttttgta aacaaatgtt gtttttggtt aaacatcttt gtgaaaacat
     1621 cctcctgtca tgtatttgct atattcaaat gttaaacccg tgcagaatag aacatataca
     1681 aaaaaaaaca acacaacaca tttttaaaca ttattaaata tcaagtattg ctggcagttc
     1741 tgtttctgtt ttacagtacc ctttgccaca gttctccgct tttcctggtc cagattccac
     1801 aagtctgatt caccaatagc aaagcgaata aacaaccaaa gcagccaatc actgcttgta
     1861 gactgtcctg cgagaccggc ccattccagc acattctgga aacttccttt atatgataat
     1921 tataaataca tttaaattat tgatacaaaa catgtaattc ctagaacata accatagcaa
     1981 tcattagttt tcagggtaat tatgtatttt taggatttga ctgcggaaag atctggtcat
     2041 gtgacgtctc atgaacgtca cggccctggg tttctataaa tacagtagga ctctcgacca
     2101 tcggcagatt tttcgaagaa gaagatcagt ttcaggagcc gtactgttcc gtttcaacgc
     2161 aaatattaac ggtaagagcg aatttcctag tttgttttca tgccattctt taaaaccata
     2221 gcgtattact ttaattatag taaactttcg ctttctttat tacaagagac gttttgtgtt
     2281 gattctccgc ggacattttc ggtcagacaa tcagaaaatg accgcggagg accagtaact
     2341 tgcattacac gtaagttaaa tcttcgtgta ttaaaatggt taggttgtta acgtcaaata
     2401 ggttaccgtg tttgcgtgtg atcaggttgg ttttgttaga tttttgtcag tatttttaat
     2461 ttatttgttt tagtttattt attttttttg ctgaatcata gtttgtgaac aaagaacccg
     2521 gatgttacat acagtacagc cgccatgtta cagagagtta taacttaatc attttaaaaa
     2581 taattttgcc ttacttttag tttgtcatgt tgagaaatga ggaaatgtta aaatgaggaa
     2641 atatccaatt aatttaatat atcaaaataa tccatgatta caatgcactg aactggagaa
     2701 aattaagatg ttttctagtg tcatgaaaca aatgtaagag atgtacattg tagatgtttt
     2761 atgtcaagaa ttggctagtt gatgcagcat actggcgata ctcagttgta ataacagtaa
     2821 cgttacatgt taatagacta ctgagtatgc tgttctgtct atgtatgctc tgtaagctac
     2881 gagaaggact tttttaaaca gtaaagggtg caatattttt acaaattgaa ttaaataaag
     2941 gctgtctatt aagtaatatg cttgatattt ttcttacttg atcggaaata agaaaaaata
     3001 taaacgttgt tgctctaaaa atcctagttc agtttagcca accacaaata cctttttgtt
     3061 cctccaacag tttttttttc ttctctataa tatttggcag tctatagtac tccaaatgtt
     3121 tccccacagt ctaactaatt ggtacagcca aaatcatgac acttattgca ataataattt
     3181 tggttcattg gcattgttga tagcctgtgc cactaatatg gtcgattgat catgcttcag
     3241 gaagaaaact atattgtttg atgtaagatt attaaatctt cacctgcttc cattacaaac
     3301 tattcccatc ttattgaatt ctggtatgtc ttaaaggatt agttcacttc ccaaatcaaa
     3361 atttacttag ccttttttca tccatgatcc ctttttttca tcattaatga agaaattgtt
     3421 tttgaaaaag tttcaagatt tttttctcta tattgtggag cttgttagtt taaaattcca
     3481 aaatgcaata tgtggcttca aatggttcta aatgatccca gtcaaggaat aacagtctta
     3541 tctaatgaaa ccattagacc tttaaaaata aaaaaaataa aagtatttat ttttaaatga
     3601 ctgagtgatt aagttgaatt tcagcgtttc cttactgtgt agaagtcctt ccttactggc
     3661 cccacccttt ggttctctgc caatctgcta cctaatgtaa tgttgtggaa cattattatt
     3721 ctttatttct taatttttta ttttttattt taaaacaatg taaactgcac agatgtgcag
     3781 tttgtttaaa atggccaatg ctttggaaat gcatgacata attagatttc atgatgcaca
     3841 aagccaaatc tcagagcttg tgcaaaatga gctatcattt cactaggtaa gaccctaaat
     3901 tttcatatag gatcatttgg acaattttgc tgcaggtaaa atgcattcta tagtccactg
     3961 tcagccattg ttttggatag tatttatttt tctctacaag tatagtcaat agttttctat
     4021 tattttaaag gtttgtaaca tttaagggtg accaaatgca aagtaaaatt tcattttcgg
     4081 gtgaactatc tcgtttaaca tgggagaagt gcaaaacata cattattggc tagaacattg
     4141 tagtattttt taaatggaaa tgtgtgattg ctaatcttac tttgaatttg tttacaggga
     4201 tccaagctta taacttcgta tagcatacat tatacgaagt tatccggtcg ccaccatggt
     4261 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     4321 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     4381 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     4441 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     4501 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     4561 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     4621 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     4681 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     4741 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     4801 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     4861 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     4921 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
     4981 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     5041 aacctcccac acctccccgt gaacgtgaaa cataaaatga atgcaattgt tgttgttaac
     5101 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5161 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     5221 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5281 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5341 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5401 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5461 accctaatca agataacttc gtatagcata cattatacga agttataccg gtggatccaa
     5521 aaagggcgaa ttcgacccaa gtttgtacaa aaaagttgaa cgagaaacgt aaaatgatat
     5581 aaatatcaat atattaaatt agattttgca taaaaaacag actacataat actgtaaaac
     5641 acaacatatg cagtcactat gaaccaacta cttagatggt attagtgacc tgtagaatta
     5701 attcgagctc tagagctgca gggcggccgc gatatcccct atagtgagtc gtattacatg
     5761 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     5821 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     5881 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     5941 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     6001 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     6061 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     6121 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     6181 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     6241 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     6301 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     6361 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     6421 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     6481 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     6541 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     6601 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     6661 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     6721 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     6781 cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6841 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6901 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6961 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7021 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7081 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7141 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7201 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7261 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7321 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7381 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7441 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
