LOCUS       sequence-344875-        5375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEST miniTol2-clone for R4-R2 3-component gateway assembly (p5E + 
            pME). Include a Multiple Cloning Site for selection marker 
            insertion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5375)
  AUTHORS   Tromp A, Wang H, Hall TE, Mowry B, Giacomotto J
  TITLE     Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new 
            gateway tools, Cre protein and guidelines.
  JOURNAL   Front Physiol. 2023 Aug 2;14:1221310. doi: 
            10.3389/fphys.2023.1221310. eCollection 2023.
  PUBMED    37601640
REFERENCE   2  (bases 1 to 5375)
  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: "Front 
            Physiol."; date: "2023-08-2"; volume: "14:1221310. doi"; pages: " 
            10.3389/fphys.2023.1221310. eCollection 2023"
COMMENT     Sequence Label: 1455_pDEST_miniTol2_R4-R2_MCS
FEATURES             Location/Qualifiers
     source          1..5375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      760..1348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1249..1268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1502..1519
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1636..1657
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1672..1677,1678..1695,1696..1702)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1710..1726
                     /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     1715..1737
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1734..1750
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1734..1750
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1768..1786
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1768..1785
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(1837..1848,1849..1986)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    2019..2142
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2167..2172,2173..2190,2191..2197)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2251..2931
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(2317..2336)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3251..3556
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3435..3454
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3597..3720)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3737..3871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3761..3780)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3815..3834
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(4072..4259,4260..4271)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4283..4302)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4284..4302)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4309..4326)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4309..4325)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4318..4340)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4466..4921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4553..4572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4763..4784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4999..5103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5104..5172,5173..5375,1..589)
                     /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     complement(5322..5341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
       61 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      121 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      181 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      241 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
      301 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      361 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      421 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      481 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
      541 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
      601 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
      661 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
      721 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
      781 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      841 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      901 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      961 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1021 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1081 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1141 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1201 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1261 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1321 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1381 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1441 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1501 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     1561 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1621 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     1681 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     1741 cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac tcaagctatg
     1801 catccaacgc gttgggagct ctcccatatg gtcgagcaga ggtgtaaaaa gtactcaaaa
     1861 attttactca agtgaaagta caagtactta gggaaaattt tactcaatta aaagtaaaag
     1921 tatctggcta gaatcttact tgagtaaaag taaaaaagta ctccattaaa attgtacttg
     1981 agtattaagg aagtagggat aacagggtaa tctcgaatca actttgtata gaaaagttga
     2041 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     2101 gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc attaggcacc
     2161 ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg
     2221 tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     2281 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     2341 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     2401 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     2461 ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     2521 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     2581 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     2641 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     2701 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     2761 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     2821 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2881 tactgcgatg agtggcaggc ggggcgtaat ctagaggatc cggcttacta aaagccagat
     2941 aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata
     3001 cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag
     3061 cgacagctat cagttgctca aggcatatat gatgtcaata tctccggttc ggtaagcaca
     3121 accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa
     3181 gggatggctg aggtcgcccg gtttattgaa atgaacggct cctttgccga cgagaacagg
     3241 ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     3301 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     3361 cagtgcacgt ctgctgtcag ataaagtccc ccgtgaactt tacccggtgg tgcatatcgg
     3421 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     3481 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     3541 gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata
     3601 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     3661 ttaatatatt gatatttata tcattttacg tttctcgttc aactttcttg tacaaagttg
     3721 atgtaccatc gatgatgatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     3781 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     3841 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     3901 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     3961 tatggctgat tatgatcctc tagatcgtgc atgcctcgag catcaccggt ccaacctgtg
     4021 agaggccgcg gttagatcta ttaccctgtt atccctaggg aaaatagaat gaagtgatct
     4081 ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt
     4141 ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt actcaagtaa
     4201 agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta ctcaagtact
     4261 ttacacctct gggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
     4321 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4381 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4441 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4501 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4561 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4621 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4681 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4741 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4801 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4861 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4921 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atcaggtggc
     4981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5101 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5161 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5221 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5281 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5341 tcccgtattg acgccgggca agagcaactc ggtcg
//
