LOCUS       sequence-90224-e        7634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tol2 transgenesis for biotin based TRAP - Avi tagged Ribosome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7634)
  AUTHORS   Housley MP, Reischauer S, Dieu M, Raes M, Stainier DY, Vanhollebeke 
            B
  TITLE     Translational profiling through biotinylation of tagged ribosomes in
            zebrafish.
  JOURNAL   Development. 2014 Oct;141(20):3988-93. doi: 10.1242/dev.111849. Epub
            2014 Sep 17.
  PUBMED    25231762
REFERENCE   2  (bases 1 to 7634)
  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"; doi: 
            "10.1242/dev.111849"; journalName: "Development"; date: "2014-10"; 
            volume: "141"; issue: "20"; pages: "3988-93"
COMMENT     Sequence Label: pT2KXIGdeltaIn-MCS-Avi-EGFP-rpl10a
FEATURES             Location/Qualifiers
     source          1..7634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(2534..2545,2546..2708)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     CDS             3127..3171
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             join(3181..3183,3184..3894)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3181..3183,3184..3894)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3223..3244)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3484..3503)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3831..3852
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             3969..3977
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4591..4725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4615..4634)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4669..4688
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(5164..5351,5352..5363)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5397..5413)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5397..5413)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5450..5470)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5450..5468)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5489..5505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5489..5505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5502..5524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5513..5529
                     /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(5537..5543,5544..5561,5562..5567))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5582..5603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5720..5737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5891..6479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5971..5990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6650..7441,7442..7510))
                     /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     7273..7292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7511..7615)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccaaatagta ggaattaccc acctgtacaa gtgctgaaaa cttggatgaa taagcccgtt
      721 tgccattttc tactgctatt ttaaatcttt tctgtatttg tctgcatttg tctttaccct
      781 tgaataaatg ttttagttgt tttttttcaa ttatgactgt gtttaacaga cattattaca
      841 ataatatgta atagtagtac acactattat tatgtaatag tacttgttga ctgtatttga
      901 gaactggacc gagtgagtgt tacgtcaccc attcaaaatg acttacttct ggctccaaca
      961 aaatgaagtt aattcagttg ccatttttca ctgtatggac atcgccgtgt tggagctaga
     1021 cgtcgccaat aagcaagaaa gagccgagat gcgtcgagtc tgagtcaccg ttcctatggc
     1081 aacccctcta accaatcaga agtaagcttg ttggaagtcc acagcctacc acttgaaagc
     1141 gggctgcaca aaatctgtca aacgttttga acgttggatg tgagagcaca tacttttatt
     1201 aaggcatctg gttggtcagt ttataatatc aacaacttgg gctacagaaa agaaaagtta
     1261 ttacagaaat tatgattaac aagtacatgt taaataaaga ttttaatatg aatgccacca
     1321 ctggagcatt catgccattt ggagcttctt cctgtttgga tcactagaag gaggaggtca
     1381 ctcattacag ttctcatata cagtcgttgg ttggttggtt ggttggttgg ttggtagatt
     1441 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt
     1501 gattgattga ttgattgatt gattgattga ttgattgatt ggtagtcaaa ataagaaata
     1561 atttccacag attcattaca gaaatgatta aatgcataca taaaaaactg gggggggggg
     1621 gatacaacaa cacacttaag tcacatttgc ctacgtaaag aaaagtaaag aaaatcaata
     1681 gctatatttt acatctcctt tttttgctgt cttttaatta gccttgtttt gctgttatct
     1741 tgattgaaac tgtaacttct tcacctgctt cttttctttg taggttttgc cagcccagaa
     1801 acaggcatgg ctgtgcaagg ccagagcacc atgcacaatg cgctgcatgt cttcatgaac
     1861 ggctcaatgt cctcagtcca gggctcagcc aacgacccca tcttcctcct tcaccatgct
     1921 ttcattgaca ggtaacaaac acgtcatgac attagactgc acagtttttg acaaagttca
     1981 tacaatctgt tgtttatagc tgctacaatt agtgaagttt gtgaatgtac ttggatgagc
     2041 agcgaaagat caattgagat caattgttag agtttggttg ccctgcagag caaagaacaa
     2101 aaaataatct ggtggcttta ctgcgtgagg ttattattgg tggaatagaa acacaaaaca
     2161 taattgcatt tatttgttta attttttatc ttatcttaac tttcatcttg catatttgtt
     2221 tcttacatca tttctagcat ctttgagcgc tggctaagaa ctcatcagcc tccccggtcc
     2281 atctacccac gtaccaatgc accaattggc cacaatgacg gctactacat ggtgccattc
     2341 cttcctcttt ataggaatgg agactacctc ctgtccaaca aggctcttgg atacgagtac
     2401 gcctacctgt tggacccagg tcattgcaca acaccagaaa tgccctctga tctgcaaaag
     2461 acgtgaatat ctgttcagac acccatatcc actctgttcc acacaggtca gaggtttgtc
     2521 caggagttct tgacagaggt gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa
     2581 gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa tcttacttga
     2641 gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag taaaagtaaa
     2701 agcaagaaag aaaactagag attcttgttt aagcttttaa tctcaaaaaa cattaaatga
     2761 aatgcataca aggttttatc ctgctttaga actgtttgta tttaattatc aaactataag
     2821 acagacaatc taatgccagt acacgctact caaagttgta aaacctcaga tttaacttca
     2881 gtagaagctg attctcaaaa ttgttagtgt caagcctagc tcttttgggg ctgaaaagca
     2941 atcctgcagt gctgaaaagc ctctcacagg cagccgatgc gggaagaggt gtattagtct
     3001 tgatagagag gctgcaaata gcaggaaacg tgagcagaga ctccctggtg tctgaaacac
     3061 aggccagatg ggccctcgag ttaattaaag gcctggccgg ccgaattcgg atccgccacc
     3121 atgtcaggac tgaacgatat cttcgaggct cagaaaatcg agtggcacga gggcgcgcca
     3181 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3241 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     3301 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3361 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3421 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3481 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3541 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3601 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3661 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3721 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3781 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3841 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccggc
     3901 cggactcaga tctcgagctc aagcttcgaa ttcagcaagg tctcgagaga cacgttgtac
     3961 gaggccgtta aggaggtcca ggccggttcc cgtcgcaaga agagaaagtt tcttgaaacc
     4021 gtggaactcc agatcagctt gaagaactac gaccctcaga aggacaagcg tttctcaggc
     4081 actgtcaggc tgaagaccac cccgcggccc aagttctctg tctgcgttct tggagaccag
     4141 cagcattgtg atgaagcaaa ggccgctgaa ctgccacaca tggacattga ggctcttaag
     4201 aagctcaata agaacaagaa gctggtcaag aagctggcaa agaagtacga tgctttcctg
     4261 gcctctgagt ctctgatcaa gcagattcct cgtatcctgg gccctggcct caacaaggca
     4321 ggaaagtttc cctctctgct cacccataat gagaacctgg gcaccaaggt ggatgaggtg
     4381 aaatccacca tcaaattcca gatgaagaag gttctgtgtc tggctgtggc cgtgggtcat
     4441 gtgaagatga gtgaggaaga gctggtctac aacatccacc tggctgtcaa cttcctggtg
     4501 tctctgctga agaagaactg gcaaaacgtc agagctcttt acatcaagag caccatggga
     4561 aaaccacagc gcctctacta gatcgatgat gatccagaca tgataagata cattgatgag
     4621 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4681 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4741 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4801 ctctacaaat gtggtatggc tgattatgat cctctagatc agatctgcga agatacggcc
     4861 acgggtgctc ttgatcctgt ggctgatttt ggactgtgct gctcgcagct gctgatgaat
     4921 cacatacttc ctccattttc ttccactgat tgactgttat aatttcccta atttccaggt
     4981 caaggtgctg tgcattgtgg taatagatgt gacatgacgt cacttccaaa ggaccaatga
     5041 acatgtctga ccaatttcat ataatgtgaa aacgattttc ataggcagaa taaataacat
     5101 ttaaattaaa ctgggcatca gcgcaattca attggtttgg taatagcaag ggaaaataga
     5161 atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa
     5221 aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt
     5281 gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat
     5341 tactcaagta ctttacacct ctggttcttg accccctacc ttcagcaagc ccagcagatc
     5401 cactagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     5461 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5521 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5581 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5641 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5701 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5761 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5821 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5881 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5941 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6001 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6061 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6121 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6181 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6241 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6301 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     6361 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6421 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6481 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6541 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6601 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6661 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6721 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6781 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6841 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6901 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6961 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7021 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7081 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7141 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7201 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7261 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7321 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7381 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7441 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7501 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7621 ttccccgaaa agtg
//
