LOCUS       sequence-90221-e        7903 bp    DNA     circular SYN 15-APR-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 7903)
  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 7903)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-EGFP
FEATURES             Location/Qualifiers
     source          1..7903
                     /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"
     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             join(4096..4098,4099..4101,4102..4815)
                     /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             join(4096..4098,4099..4101,4102..4812)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4141..4162)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4402..4421)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4749..4770
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4860..4994
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4884..4903)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4938..4957
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5666..5682)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5719..5739)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5719..5737)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5758..5774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5758..5774)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5771..5793)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5782..5798
                     /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(5806..5812,5813..5830,5831..5836))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5851..5872
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5989..6006)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6160..6748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6240..6259)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6919..7710,7711..7779))
                     /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     7542..7561
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7780..7884)
                     /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 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4141 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4201 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     4261 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     4321 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4381 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4441 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4501 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4561 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4621 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4681 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4741 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     4801 gagctgtaca agtaaagcgg ccgccaccgc ggtggagctc gaattaattc atcgatgatg
     4861 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     4921 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     4981 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     5041 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc
     5101 ctctagatca gatctgcgaa gatacggcca cgggtgctct tgatcctgtg gctgattttg
     5161 gactgtgctg ctcgcagctg ctgatgaatc acatacttcc tccattttct tccactgatt
     5221 gactgttata atttccctaa tttccaggtc aaggtgctgt gcattgtggt aatagatgtg
     5281 acatgacgtc acttccaaag gaccaatgaa catgtctgac caatttcata taatgtgaaa
     5341 acgattttca taggcagaat aaataacatt taaattaaac tgggcatcag cgcaattcaa
     5401 ttggtttggt aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt
     5461 tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta
     5521 agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat
     5581 aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga
     5641 ccccctacct tcagcaagcc cagcagatcc actagttcta gagcggccgc caccgcggtg
     5701 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     5761 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5821 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5881 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5941 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6001 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6061 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6121 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6181 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6241 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6301 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6361 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6421 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6481 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6541 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6601 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6661 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6721 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6781 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6841 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6901 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6961 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7021 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7081 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7141 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7201 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7261 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7321 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7381 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7441 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7501 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7561 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7621 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7681 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7741 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7801 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7861 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtg
//
