LOCUS       sequence-90222-e        7883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tol2 transgenesis for biotin based TRAP - BirA ligase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7883)
  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 7883)
  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-birA-tagRFP
FEATURES             Location/Qualifiers
     source          1..7883
                     /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             3121..4083
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3121..4083
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     regulatory      4090..4099
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4096..4827
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     polyA_signal    4840..4974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4864..4883)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4918..4937
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(5413..5600,5601..5612)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5646..5662)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5646..5662)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5699..5719)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5699..5717)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5738..5754)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5738..5754)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5751..5773)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5762..5778
                     /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(5786..5792,5793..5810,5811..5816))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5831..5852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5969..5986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6140..6728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6220..6239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6899..7690,7691..7759))
                     /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     7522..7541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7760..7864)
                     /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 atgaaggata acaccgtgcc actgaaattg attgccctgt tagcgaacgg tgaatttcac
     3181 tctggcgagc agttgggtga aacgctggga atgagccggg cggctattaa taaacacatt
     3241 cagacactgc gtgactgggg cgttgatgtc tttaccgttc cgggtaaagg atacagcctg
     3301 cctgagccta tccagttact taatgctaaa cagatattgg gtcagctgga tggcggtagt
     3361 gtagccgtgc tgcctgtgat tgactccacg aatcagtacc ttcttgatcg tatcggagag
     3421 cttaaatcgg gcgatgcttg cattgcagaa taccagcagg ctggccgtgg tcgccggggt
     3481 cggaaatggt tttcgccttt tggcgcaaac ttatatttgt cgatgttctg gcgtctggaa
     3541 caaggcccgg cggcggcgat tggtttaagt ctggttatcg gtatcgtgat ggcggaagta
     3601 ttacgcaagc tgggtgcaga taaagttcgt gttaaatggc ctaatgacct ctatctgcag
     3661 gatcgcaagc tggcaggcat tctggtggag ctgactggca aaactggcga tgcggcgcaa
     3721 atagtcattg gagccgggat caacatggca atgcgccgtg ttgaagagag tgtcgttaat
     3781 caggggtgga tcacgctgca ggaagcgggg atcaatctcg atcgtaatac gttggcggcc
     3841 atgctaatac gtgaattacg tgctgcgttg gaactcttcg aacaagaagg attggcacct
     3901 tatctgtcgc gctgggaaaa gctggataat tttattaatc gcccagtgaa acttatcatt
     3961 ggtgataaag aaatatttgg catttcacgc ggaatagaca aacagggggc tttattactt
     4021 gagcaggatg gaataataaa accctggatg ggcggtgaaa tatccctgcg tagtgcagaa
     4081 aaaaagcttg ccaccatggt gtctaagggc gaagagctga ttaaggagaa catgcacatg
     4141 aagctgtaca tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa
     4201 ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc
     4261 cccttcgcct tcgacatcct ggctaccagc ttcatgtacg gcagcagaac cttcatcaac
     4321 cacacccagg gcatccccga tttctttaag cagtccttcc ctgagggctt cacatgggag
     4381 agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag
     4441 gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcccatc caacggccct
     4501 gtgatgcaga agaaaacact cggctgggag gccaacaccg agatgctgta ccccgctgac
     4561 ggcggcctgg aaggcagaac cgacatggcc ctgaagctcg tgggcggggg ccacctgatc
     4621 tgcaacttca agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc
     4681 gtctactatg tggaccacag actggaaaga atcaaggagg ccgacaaaga gacctacgtc
     4741 gagcagcacg aggtggctgt ggccagatac tgcgacctcc ctagcaaact ggggcacaaa
     4801 cttaatggca tggacgagct gtacaagtaa atcgatgatg atccagacat gataagatac
     4861 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     4921 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     4981 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     5041 aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctagatca gatctgcgaa
     5101 gatacggcca cgggtgctct tgatcctgtg gctgattttg gactgtgctg ctcgcagctg
     5161 ctgatgaatc acatacttcc tccattttct tccactgatt gactgttata atttccctaa
     5221 tttccaggtc aaggtgctgt gcattgtggt aatagatgtg acatgacgtc acttccaaag
     5281 gaccaatgaa catgtctgac caatttcata taatgtgaaa acgattttca taggcagaat
     5341 aaataacatt taaattaaac tgggcatcag cgcaattcaa ttggtttggt aatagcaagg
     5401 gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa taaaattgta
     5461 aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta aaagtattga
     5521 tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag tacagtaatc
     5581 aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct tcagcaagcc
     5641 cagcagatcc actagttcta gagcggccgc caccgcggtg gagctccagc ttttgttccc
     5701 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5761 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5821 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5881 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5941 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6001 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6061 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6121 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6181 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6241 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6301 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6361 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6421 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6481 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6541 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6601 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6661 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6721 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6781 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6841 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6901 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6961 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7021 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7081 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7141 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7201 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7261 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7321 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7381 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7441 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7501 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7561 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7621 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7681 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7741 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7801 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7861 cgcgcacatt tccccgaaaa gtg
//
