LOCUS       sequence-344874-        7071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEST miniTol2-clone for R4-R2 3-component gateway assembly (p5E + 
            pME). Include a eGFP selection marker (Crystallin promoter 
            Lens/Eyes).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7071)
  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 7071)
  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: 1456_pDEST_miniTol2_R4-R2_Cryst-eGFP
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      500..509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1274..1408
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1425..1548
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1589..1894)
                     /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(1691..1710)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2214..2894)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     2809..2828
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2948..2954,2955..2972,2973..2978))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3003..3126)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(3159..3296,3297..3308)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(3359..3377)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3360..3377)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3395..3411)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3395..3411)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3408..3430)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3419..3435
                     /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(3443..3449,3450..3467,3468..3473))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3488..3509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3626..3643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3797..4385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3877..3896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4556..5347,5348..5416))
                     /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     5179..5198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5417..5521)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5599..6054)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5736..5757)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5948..5967
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6180..6202
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6194..6211
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6195..6211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6218..6237
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6218..6236
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(6249..6260,6261..6448)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    6611..6732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(6855..7071,1..497,498..500,501..503))
                     /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             complement(join(6858..7071,1..497,498..500,501..503))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6900..6921)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      301 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcctcgc ccttgctcac catggtggcg accggtaatg tcagacctgg taactcctta
      541 ctgtaactta ttgcacctga tgaacaaaag atgtgaactg aacgcaagta ggaggctaat
      601 agaggctagt acagtacaaa gatccccaaa tgatgtgtgc cggccttagt ggaggcagac
      661 ccctggttat atacctgtgc tgcagaaaga ggggcttaca aatggatctc tctggaatgg
      721 cattatctca tggtgggaac agtgttgtca gcagaatcag cggggtggac tctgacctga
      781 gaatgacagt ctagcactga atggctcaga caatactctt gtggccaact atcaccggaa
      841 aataagtgcc tgccaaaaca aaccagacca gatgaagcta catggctgta gaacgtcagt
      901 atttaatgtg ttgtggccac ttggttcacc agatttaaga gctaattttt ttaaatatca
      961 cagataatca atcgcttttt tttttcttga ctgaaaagct tagactggaa aattctttga
     1021 aaaagtttgc actacagaaa ataaatacat aacatcctgc actgaatgca cactattaat
     1081 agaaaagaga gcattattgt aggaaatgta ttgtatatgt acattctgtg catttcacaa
     1141 ttttctcgag gcatgcacga tctagaggat cataatcagc cataccacat ttgtagaggt
     1201 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     1261 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1321 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1381 catcaatgta tcttatcatg tctggatcat catcgatggt acatcaactt tgtacaagaa
     1441 agttgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa
     1501 aaaacagact acataatact gtaaaacaca acatatccag tcactatggt cgacctgcag
     1561 actggctgtg tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt
     1621 ttttgatgtc attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg
     1681 gcacactggc catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg
     1741 ggtaaagttc acgggggact ttatctgaca gcagacgtgc actggccagg gggatcacca
     1801 tccgtcgccc gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc
     1861 tctctctttt ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcggca
     1921 aaggagccgt tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct
     1981 ttccagcgtt cggcacgcag acgacgggct tcattctgca tggttgtgct taccgaaccg
     2041 gagatattga catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg
     2101 taatacgctg cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata
     2161 tattcttata ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa
     2221 gccggatcct ctagattacg ccccgcctgc cactcatcgc agtactgttg taattcatta
     2281 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
     2341 atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag
     2401 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
     2461 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
     2521 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
     2581 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     2641 catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg
     2701 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     2761 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     2821 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     2881 atttttttct ccattttagc ttccttagct cctgaaaatc tcgacggatc ctaactcaaa
     2941 atccacacat tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgcggcc
     3001 gccatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
     3061 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact tttctataca
     3121 aagttgattc gagattaccc tgttatccct acttccttaa tactcaagta caattttaat
     3181 ggagtacttt tttactttta ctcaagtaag attctagcca gatactttta cttttaattg
     3241 agtaaaattt tccctaagta cttgtacttt cacttgagta aaatttttga gtacttttta
     3301 cacctctgct cgaccatatg ggagagctcc caacgcgttg gatgcatagc ttgagtattc
     3361 tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3421 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3481 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3541 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3601 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3661 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3721 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3781 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3841 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3901 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3961 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4021 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4081 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4141 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4201 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4261 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4321 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4381 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4441 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4501 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4561 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4621 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4681 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4741 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4801 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4861 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4921 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4981 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5041 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5101 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5161 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5221 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5281 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5341 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5401 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5461 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5521 gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg
     5581 agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5641 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5701 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5761 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5821 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     5881 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     5941 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6001 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc
     6061 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     6121 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     6181 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga
     6241 attgggccca gaggtgtaaa gtacttgagt aattttactt gattactgta cttaagtatt
     6301 atttttgggg atttttactt tacttgagta caattaaaaa tcaatacttt tacttttact
     6361 taattacatt tttttagaaa aaaaagtact ttttactcct tacaatttta tttacagtca
     6421 aaaagtactt attttttgga gatcacttca ttctattttc cctagggata acagggtaat
     6481 agatctaacc gcggcctctc acaggcagcc gatgcgggaa gaggtgtatt agtcttgata
     6541 gagaggctgc aaatagcagg aaacgtgagc agagactccc tggtgtctga aacacaggcc
     6601 agattggcct taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     6661 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     6721 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     6781 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcta
     6841 gagtcgcggc cgctttactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
     6901 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac
     6961 tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     7021 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg g
//
