LOCUS       sequence-344872-        7063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEST miniTol2-clone for R4-R2 3-component gateway assembly (p5E + 
            pME). Include a mCherry selection marker (Crystallin promoter 
            Lens/Eyes).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7063)
  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 7063)
  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: 1457_pDEST_miniTol2_R4-R2_Cryst_mCherry
FEATURES             Location/Qualifiers
     source          1..7063
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(195..783)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(275..294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(954..1745,1746..1814))
                     /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     1577..1596
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1815..1919)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1997..2452)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2134..2155)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2346..2365
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2578..2600
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2592..2609
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2593..2609
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2616..2635
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2616..2634
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(2647..2658,2659..2846)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    3010..3131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3254..3964)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGHHSTGGMDELYK"
     primer_bind     complement(3334..3357)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3532..3551)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3801..3819
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3961..3970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4735..4869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    4886..5009
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5050..5355)
                     /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     complement(5152..5171)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5675..6355)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     6270..6289
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6409..6415,6416..6433,6434..6439))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(6464..6587)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(6620..6757,6758..6769)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(6820..6838)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6821..6838)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6856..6872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6856..6872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6869..6891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6880..6896
                     /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(6904..6910,6911..6928,6929..6934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6949..6970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
       61 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      121 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      181 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      241 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      301 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      361 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      421 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      481 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      541 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      601 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      661 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      721 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      781 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      841 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      901 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      961 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1021 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1081 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1141 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1201 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1261 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1321 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1381 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1441 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1501 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1561 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1621 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1681 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1741 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1801 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1861 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1921 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
     1981 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2041 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2101 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2161 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2221 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2281 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2341 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2401 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
     2461 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     2521 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2581 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
     2641 tgggcccaga ggtgtaaagt acttgagtaa ttttacttga ttactgtact taagtattat
     2701 ttttggggat ttttacttta cttgagtaca attaaaaatc aatactttta cttttactta
     2761 attacatttt tttagaaaaa aaagtacttt ttactcctta caattttatt tacagtcaaa
     2821 aagtacttat tttttggaga tcacttcatt ctattttccc tagggataac agggtaatag
     2881 atctaaccgc ggcctctcac aggcagccga tgcgggaaga ggtgtattag tcttgataga
     2941 gaggctgcaa atagcaggaa acgtgagcag agactccctg gtgtctgaaa cacaggccca
     3001 tttacgcctt aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3061 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3121 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     3181 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatctag
     3241 agtcgcggcc gctttacttg tacagctcgt ccatgccgcc ggtggagtgg tggccctcgg
     3301 cgcgctcgta ctgttccacg atggtgtagt cctcgttgtg ggaggtgatg tccagcttga
     3361 tgttgacgtt gtaggcgccg ggcagctgca cgggcttctt ggccttgtag gtggtcttga
     3421 cctcggcgtc gtagtggccg ccgtccttca gcttcagcct ctgcttgatc tcgcccttca
     3481 gggcgccgtc ctcggggtac atccgctcgg aggaggcctc ccagcccatg gtcttcttct
     3541 gcattacggg gccgtcggag gggaagttgg tgccgcgcag cttcaccttg tagatgaact
     3601 cgccgtcctg cagggaggag tcctgggtca cggtcaccac gccgccgtcc tcgaagttca
     3661 tcacgcgctc ccacttgaag ccctcgggga aggacagctt caagtagtcg gggatgtcgg
     3721 cggggtgctt cacgtaggcc ttggagccgt acatgaactg aggggacagg atgtcccagg
     3781 cgaagggcag ggggccgccc ttggtcacct tcagcttggc ggtctgggtg ccctcgtagg
     3841 ggcggccctc gccctcgccc tcgatctcga actcgtggcc gttcacggag ccctccatgt
     3901 gcaccttgaa gcgcatgaac tccttgatga tggccatgtt gtcatcctcg cccttgctca
     3961 ccatggtggc gaccggtaat gtcagacctg gtaactcctt actgtaactt attgcacctg
     4021 atgaacaaaa gatgtgaact gaacgcaagt aggaggctaa tagaggctag tacagtacaa
     4081 agatccccaa atgatgtgtg ccggccttag tggaggcaga cccctggtta tatacctgtg
     4141 ctgcagaaag aggggcttac aaatggatct ctctggaatg gcattatctc atggtgggaa
     4201 cagtgttgtc agcagaatca gcggggtgga ctctgacctg agaatgacag tctagcactg
     4261 aatggctcag acaatactct tgtggccaac tatcaccgga aaataagtgc ctgccaaaac
     4321 aaaccagacc agatgaagct acatggctgt agaacgtcag tatttaatgt gttgtggcca
     4381 cttggttcac cagatttaag agctaatttt tttaaatatc acagataatc aatcgctttt
     4441 ttttttcttg actgaaaagc ttagactgga aaattctttg aaaaagtttg cactacagaa
     4501 aataaataca taacatcctg cactgaatgc acactattaa tagaaaagag agcattattg
     4561 taggaaatgt attgtatatg tacattctgt gcatttcaca attttctcga ggcatgcacg
     4621 atctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     4681 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     4741 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4801 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4861 gtctggatca tcatcgatgg tacatcaact ttgtacaaga aagttgaacg agaaacgtaa
     4921 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     4981 tgtaaaacac aacatatcca gtcactatgg tcgacctgca gactggctgt gtataaggga
     5041 gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg
     5101 gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt
     5161 ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggggac
     5221 tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc
     5281 aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta
     5341 aaccttaaac tgcatttcac cagcccctgt tctcgtcggc aaaggagccg ttcatttcaa
     5401 taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca
     5461 gacgacgggc ttcattctgc atggttgtgc ttaccgaacc ggagatattg acatcatata
     5521 tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc
     5581 atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa
     5641 tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc tctagattac
     5701 gccccgcctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
     5761 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
     5821 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
     5881 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa
     5941 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
     6001 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
     6061 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
     6121 ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
     6181 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
     6241 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
     6301 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag
     6361 cttccttagc tcctgaaaat ctcgacggat cctaactcaa aatccacaca ttatacgagc
     6421 cggaagcata aagtgtaaag cctggggtgc ctaatgcggc cgccatagtg actggatatg
     6481 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
     6541 atttatatca ttttacgttt ctcgttcaac ttttctatac aaagttgatt cgagattacc
     6601 ctgttatccc tacttcctta atactcaagt acaattttaa tggagtactt ttttactttt
     6661 actcaagtaa gattctagcc agatactttt acttttaatt gagtaaaatt ttccctaagt
     6721 acttgtactt tcacttgagt aaaatttttg agtacttttt acacctctgc tcgaccatat
     6781 gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag
     6841 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6901 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6961 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     7021 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttg
//
