LOCUS       sequence-344833-        7484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p5E tol2kit compatible clone encoding a Ubiquitin promoter and a 
            tagBFP sequence terminated by 3x polyA sequences and flanked by 
            floxP sites for CRE-excision.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7484)
  AUTHORS   Guo G, Tromp A, Wang H, Hall TE, Giacomotto J
  TITLE     Cre-Lox miRNA-delivery technology optimized for inducible microRNA 
            and gene-silencing studies in zebrafish
  JOURNAL   bioRxiv 2024.02.22.581357
REFERENCE   2  (bases 1 to 7484)
  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: "bioRxiv 
            2024.02.22.581357"
COMMENT     Sequence Label: 50_p5E_UBI_loxTagBFP
FEATURES             Location/Qualifiers
     source          1..7484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(174..201)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(293..379)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     428..450
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     442..459
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     443..459
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    499..594
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(4117..4150)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4163..4864
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4992..5113
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5029..5048)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5083..5102
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5257..5278)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5367..5400)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5432..5555
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5631..5650)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5632..5650)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5655..5671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5655..5671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5773..5792)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5784..6593
                     /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(5851..5870)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6740..7328
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7229..7248
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7482..7484,1..15)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
       61 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
      121 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
      181 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
      241 cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc
      301 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact
      361 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcgttaac gctagcatgg
      421 atgttttccc agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg gcccgagtta
      481 acgctaccat ggagctccaa ataatgattt tattttgact gatagtgacc tgttcgttgc
      541 aacaaattga taagcaatgc ttttttataa tgccaacttt gtatagaaaa gttggctccg
      601 aattcgccct taaaactcga gaccagcaaa gttctagaat ttgtcgaaac atttatgtta
      661 tatatttcct gaaaaaaatt ctgagtaagt tcttaagtgt tattgccagc aacataaaca
      721 acagacggca aaatgaataa atgataacaa agcagtaggc ttaaataaac ctaattttta
      781 taggctgttc tctacaaccc tcaaacagtg attagttttg tacttataaa cttgcccttt
      841 cattcatatt tcaagaaaat tggttcagaa gatctggata ttctagcagt tgttcaagct
      901 catggaggga tcagtgacct gattccacaa tgactaggcc taatccagaa attagatgac
      961 tgtcaacata aaaaggcaca gcactcacta gctgccctat atattttatt atattttaca
     1021 tatattattt tatttattta gctctgagtg ctgtactttc tggttaaaga aaactgctta
     1081 caacagctaa cctgtactac ctcaggctca gggaatttgg aacaggtttg tctggtttgt
     1141 ttctttaacc atgcatgctt gttttcaact atggcaacac agtcacatgg gacattacag
     1201 aaatgatttg tcgatgacat gcgacttttc tttaataaag cgcaaagatc ccaaaaagca
     1261 aacttttaac aaaaatcata taattatatt ttcaatccag ctttgtagca actttgtgct
     1321 gctgttcact cagcaacaga tagtcagtat aaggtcagtg tgtctcaaag cagtgccatc
     1381 tgtttcacac attgcgttct atatataagt gtgctggttg acacgacact gtataaggcc
     1441 taggctaaaa cacaaacaat gtagaatgac actgtgtttt ttttgtaaac aaatgttgtt
     1501 tttggttaaa catctttgtg aaaacatcct cctgtcatgt atttgctata ttcaaatgtt
     1561 aaacccgtgc agaatagaac atatacaaaa aaaaacaaca caacacattt ttaaacatta
     1621 ttaaatatca agtattgctg gcagttctgt ttctgtttta cagtaccctt tgccacagtt
     1681 ctccgctttt cctggtccag attccacaag tctgattcac caatagcaaa gcgaataaac
     1741 aaccaaagca gccaatcact gcttgtagac tgtcctgcga gaccggccca ttccagcaca
     1801 ttctggaaac ttcctttata tgataattat aaatacattt aaattattga tacaaaacat
     1861 gtaattccta gaacataacc atagcaatca ttagttttca gggtaattat gtatttttag
     1921 gatttgactg cggaaagatc tggtcatgtg acgtctcatg aacgtcacgg ccctgggttt
     1981 ctataaatac agtaggactc tcgaccatcg gcagattttt cgaagaagaa gatcagtttc
     2041 aggagccgta ctgttccgtt tcaacgcaaa tattaacggt aagagcgaat ttcctagttt
     2101 gttttcatgc cattctttaa aaccatagcg tattacttta attatagtaa actttcgctt
     2161 tctttattac aagagacgtt ttgtgttgat tctccgcgga cattttcggt cagacaatca
     2221 gaaaatgacc gcggaggacc agtaacttgc attacacgta agttaaatct tcgtgtatta
     2281 aaatggttag gttgttaacg tcaaataggt taccgtgttt gcgtgtgatc aggttggttt
     2341 tgttagattt ttgtcagtat ttttaattta tttgttttag tttatttatt ttttttgctg
     2401 aatcatagtt tgtgaacaaa gaacccggat gttacataca gtacagccgc catgttacag
     2461 agagttataa cttaatcatt ttaaaaataa ttttgcctta cttttagttt gtcatgttga
     2521 gaaatgagga aatgttaaaa tgaggaaata tccaattaat ttaatatatc aaaataatcc
     2581 atgattacaa tgcactgaac tggagaaaat taagatgttt tctagtgtca tgaaacaaat
     2641 gtaagagatg tacattgtag atgttttatg tcaagaattg gctagttgat gcagcatact
     2701 ggcgatactc agttgtaata acagtaacgt tacatgttaa tagactactg agtatgctgt
     2761 tctgtctatg tatgctctgt aagctacgag aaggactttt ttaaacagta aagggtgcaa
     2821 tatttttaca aattgaatta aataaaggct gtctattaag taatatgctt gatatttttc
     2881 ttacttgatc ggaaataaga aaaaatataa acgttgttgc tctaaaaatc ctagttcagt
     2941 ttagccaacc acaaatacct ttttgttcct ccaacagttt ttttttcttc tctataatat
     3001 ttggcagtct atagtactcc aaatgtttcc ccacagtcta actaattggt acagccaaaa
     3061 tcatgacact tattgcaata ataattttgg ttcattggca ttgttgatag cctgtgccac
     3121 taatatggtc gattgatcat gcttcaggaa gaaaactata ttgtttgatg taagattatt
     3181 aaatcttcac ctgcttccat tacaaactat tcccatctta ttgaattctg gtatgtctta
     3241 aaggattagt tcacttccca aatcaaaatt tacttagcct tttttcatcc atgatccctt
     3301 tttttcatca ttaatgaaga aattgttttt gaaaaagttt caagattttt ttctctatat
     3361 tgtggagctt gttagtttaa aattccaaaa tgcaatatgt ggcttcaaat ggttctaaat
     3421 gatcccagtc aaggaataac agtcttatct aatgaaacca ttagaccttt aaaaataaaa
     3481 aaaataaaag tatttatttt taaatgactg agtgattaag ttgaatttca gcgtttcctt
     3541 actgtgtaga agtccttcct tactggcccc accctttggt tctctgccaa tctgctacct
     3601 aatgtaatgt tgtggaacat tattattctt tatttcttaa ttttttattt tttattttaa
     3661 aacaatgtaa actgcacaga tgtgcagttt gtttaaaatg gccaatgctt tggaaatgca
     3721 tgacataatt agatttcatg atgcacaaag ccaaatctca gagcttgtgc aaaatgagct
     3781 atcatttcac taggtaagac cctaaatttt catataggat catttggaca attttgctgc
     3841 aggtaaaatg cattctatag tccactgtca gccattgttt tggatagtat ttatttttct
     3901 ctacaagtat agtcaatagt tttctattat tttaaaggtt tgtaacattt aagggtgacc
     3961 aaatgcaaag taaaatttca ttttcgggtg aactatctcg tttaacatgg gagaagtgca
     4021 aaacatacat tattggctag aacattgtag tattttttaa atggaaatgt gtgattgcta
     4081 atcttacttt gaatttgttt acagggatcc aagcttataa cttcgtatag catacattat
     4141 acgaagttat ccggtcgcca ccatgagcga gctgattaag gagaacatgc acatgaagct
     4201 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     4261 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     4321 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     4381 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     4441 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     4501 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     4561 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     4621 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     4681 catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     4741 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     4801 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
     4861 ttaaagcggc cgcggtacca ctctagatca taatcagcca taccacattt gtagaggttt
     4921 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     4981 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5041 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5101 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5161 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5221 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5281 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5341 ctacgtgaac catcacccta atcaagataa cttcgtatag catacattat acgaagttat
     5401 accggtggat cctttaaggg cgaattcgac ccaagtttgt acaaaaaagt tgaacgagaa
     5461 acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa acagactaca
     5521 taatactgta aaacacaaca tatgcagtca ctatgaacca actacttaga tggtattagt
     5581 gacctgtaga attaattcga gctctagagc tgcagggcgg ccgcgatatc ccctatagtg
     5641 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     5701 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     5761 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat
     5821 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     5881 aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga
     5941 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     6001 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     6061 ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg
     6121 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     6181 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     6241 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     6301 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     6361 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     6421 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     6481 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     6541 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     6601 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct
     6661 catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag
     6721 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6781 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6841 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6901 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6961 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7021 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7081 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7141 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7201 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7261 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7321 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7381 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7441 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagc
//
