LOCUS       sequence-152595-        7221 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Myl7 promoter driving HA-tagged biotin ligase (BirA) with NLS, a 
            Thosea asigna virus peptide (2A), and membrane-tethered mCherry 
            protein (membCherry); flanked by Tol2 sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7221)
  AUTHORS   Trinh LA, Chong-Morrison V, Gavriouchkina D, Hochgreb-Hagele T, 
            Senanayake U, Fraser SE, Sauka-Spengler T
  TITLE     Biotagging of Specific Cell Populations in Zebrafish Reveals Gene 
            Regulatory Logic Encoded in the Nuclear Transcriptome.
  JOURNAL   Cell Rep. 2017 Apr 11;19(2):425-440. doi: 
            10.1016/j.celrep.2017.03.045.
  PUBMED    28402863
REFERENCE   2  (bases 1 to 7221)
  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: 
            "10.1016/j.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-11- 11"; volume: "19"; issue: "2"; pages: "425-440"
COMMENT     Sequence Label: pMT-myl7-NLS-BirA-2A-mCherry_Ras
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..19
                     /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)."
     primer_bind     8..30
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     27..43
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..43
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     62..82
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        64..82
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     119..135
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     119..135
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      2034..2043
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2049..2075
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             2082..2108
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             2115..2141
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             2157..2177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2184..3146
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2184..3146
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3162..3215
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3222..3929
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3367..3385)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3635..3654
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(4042..4060)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    4067..4201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4091..4110)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4145..4164
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(4471..4489)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4499..4516)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4499..4515)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4508..4530)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4657..5112
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4744..4763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4954..4975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5138..5242
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5243..5311,5312..6103)
                     /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     complement(5461..5480)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6274..6862
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6763..6782
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7016..7033
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7150..7171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7186..7191,7192..7209,7210..7216)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
       61 cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc
      121 tagaactagt ggatctgctg ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa
      181 gtacttgagt aattttactt gattactgta cttaagtatt atttttgggg atttttactt
      241 tacttgagta caattaaaaa tcaatacttt tacttttact taattacatt tttttagaaa
      301 aaaaagtact ttttactcct tacaatttta tttacagtca aaaagtactt attttttgga
      361 gatcacttgg gcccggctcg agtgtgacca aagcttaaat cagttgtgtt aaataagaga
      421 cattcaaaat aaatgtaaat gagctctcca aatcagcaga cttaacattc tttaaaatga
      481 ttgattcaat agtgatacaa atcaggcata gcagttgtaa cttttgataa attacagaaa
      541 atgtcaaata cggagaaccg attctttttt atgacacatc caagcacaca tttaacacaa
      601 tccaggcaaa ccccgaattt cagagtcaca agcactgttt gtacaagagc tttgcctaag
      661 gacgcacagt ctctatgagt ccaggtcgtt ggtttcactc ttattttaaa catgtgacat
      721 ttttcctgcc atcctgtctt aggctgctgt ttgcttcatt tcatgtcaca ttaatttcct
      781 cagtagcacc ttttacacac acagccaatc ttttccagaa cattcaattg ctttgaagag
      841 ataatgtgtg aacaaatcca tttagaaaag gaaaattaag aatttgtaaa atcatctgta
      901 aattgttggc attcttctgt atatgaacat cacatcattt acaggtaaag gtctggtcat
      961 taattatatg acaatttact ggtattattt tgtgaaaggg gctattttca atgcattcat
     1021 ccatcctttt catccctcaa atctctcatt cacgtccccc tccccatctg cacactttat
     1081 ctcattttcc accctgctgg aatctgagca cttgtgcagt tatcagggct cctgtattta
     1141 ggaggctctg ggtgtccatg taggggacga acagaaacac tgcagacctt tatagaagaa
     1201 caaatgataa gagtcctcat acataaagac tccattagaa acgtcagtga cccaggagcc
     1261 cagaccaaca gcaaagcaga cagtgaacgt ggtgagtaga caaagctata cttttttggt
     1321 tttgaatata atattaatgt gaaaataaaa agggtctata tgaagttaaa tggtgtttgt
     1381 ttgttgatat taaatattag aagcatcttt tctgcatttg tatgttgtga ttctatcttt
     1441 taatggtatt tttacttagg tacttttttc tgtacactgt aaaacgcaaa aaagttaagg
     1501 taactcaaat catttgagaa aaccgattgt aacaagccat ctgaagttca aaaactaatc
     1561 taaatgagta ctgtgaactt aatctatttg agtaaaagaa gcaatttgag cacagtaaaa
     1621 cccagtaaat gaagagaact caaccaactg agtactgtaa aacccaataa gttgaggcag
     1681 ctcacaccgt tagaggaaac cgattgctac aaaccatttg agttaaaaaa aagaatctat
     1741 atgagtactg tgaacttact ccatttaagt tgaagtaatg aggtaattaa ttaactcatt
     1801 accctcaaca ctaagttcaa aactcttttc aaatgagtag aattaatctt cagccaattt
     1861 tgagttaact atactcattt catttgataa agttgactat tgggttttac agtgtataaa
     1921 tgctataaat tgagtttcca aataatgctt ctcttttttt tttactcaca ggctagtcga
     1981 attcaaggtt aagcggccgc aagctagatt ggcattccgg tactgttggt aaagccacca
     2041 tggccaccta tgacgtccca gattacgcaa gtttgcccgg gtatgatgtt cctgattatg
     2101 ctagcctccc gggttacgat gtgcccgact atgcctcact tccaggcaga tctcctccaa
     2161 aaaagaagag aaaggtacga tctatgaagg ataacaccgt gccactgaaa ttgattgccc
     2221 tgttagcgaa cggtgaattt cactctggcg agcagttggg tgaaacgctg ggaatgagcc
     2281 gggcggctat taataaacac attcagacac tgcgtgactg gggcgttgat gtctttaccg
     2341 ttccgggtaa aggatacagc ctgcctgagc ctatccagtt acttaatgct aaacagatat
     2401 tgggtcagct ggatggcggt agtgtagccg tgctgccagt gattgactcc acgaatcagt
     2461 accttcttga tcgtatcgga gagcttaaat cgggcgatgc ttgcattgca gaataccagc
     2521 aggctggccg tggtcgccgg ggtcggaaat ggttttcgcc ttttggcgca aacttatatt
     2581 tgtcgatgtt ctggcgtctg gaacaaggcc cggcggcggc gattggttta agtctggtta
     2641 tcggtatcgt gatggcggaa gtattacgca agctgggtgc agataaagtt cgtgttaaat
     2701 ggcctaatga cctctatctg caggatcgca agctggcagg cattctggtg gagctgactg
     2761 gcaaaactgg cgatgcggcg caaatagtca ttggagccgg gatcaacatg gcaatgcgcc
     2821 gtgttgaaga gagtgtcgtt aatcaggggt ggatcacgct gcaggaagcg gggatcaatc
     2881 tcgatcgtaa tacgttggcg gccatgctaa tacgtgaatt acgtgctgcg ttggaactct
     2941 tcgaacaaga aggattggca ccttatctgt cgcgctggga aaagctggat aattttatta
     3001 atcgcccagt gaaacttatc attggtgata aagaaatatt tggcatttca cgcggaatag
     3061 acaaacaggg ggctttatta cttgagcagg atggaataat aaaaccctgg atgggcggtg
     3121 aaatatccct gcgtagtgca gaaaaagata tcggatcagg agagggcagg ggaagtcttc
     3181 taacatgcgg ggacgtggag gaaaatcccg ggcccagatc tatggtgagc aagggcgagg
     3241 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     3301 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     3361 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     3421 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     3481 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     3541 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     3601 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     3661 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     3721 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     3781 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     3841 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     3901 ccaccggcgg catggacgag ctgtacaagg gaggaggaag atctaagctg aaccctcctg
     3961 atgagagtgg ccccggctgc atgagctgca agtgtgtgct ctcctgatct agaactatag
     4021 tgagtcgtat tacccggact ccctatagtg agtcgtatta accggtgatc cagacatgat
     4081 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4141 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4201 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     4261 ttgatatccc gggtttaaac gcatgcagat ctaatactca agtacaattt taatggagta
     4321 cttttttact tttactcaag taagattcta gccagatact tttactttta attgagtaaa
     4381 attttcccta agtacttgta ctttcacttg agtaaaattt ttgagtactt tttacacctc
     4441 tgtcaagaac catatgggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac
     4501 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     4561 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     4621 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     4681 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4741 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4801 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4861 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4921 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4981 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5041 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5101 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5161 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5221 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5281 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5341 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5401 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5461 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5521 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5581 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5641 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5701 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5761 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5821 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5881 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5941 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6001 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6061 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6121 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6181 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6241 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6301 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6361 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6421 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6481 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6541 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6601 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6661 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6721 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6781 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6841 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6901 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6961 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7021 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     7081 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     7141 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     7201 tccggctcgt atgttgtgtg g
//
