LOCUS       sequence-152592-        5697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCS for cloning promoter to drive HA-tagged biotin ligase (BirA), 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 5697)
  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 5697)
  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: 
            "10.1016/j.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-11- 11"; volume: "19"; issue: "2"; pages: "425-440"
COMMENT     Sequence Label: pMT-MCS-cytoBirA-2A-mCherry_Ras
FEATURES             Location/Qualifiers
     source          1..5697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      353..941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     842..861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1095..1112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1229..1250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1265..1270,1271..1288,1289..1295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1303..1319
                     /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     1308..1330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1327..1343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1327..1343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1362..1382
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1364..1382
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1419..1435
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1419..1435
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(1469..1480,1481..1668)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        1752..1770
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1752..1769
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1832..1841
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1847..1873
                     /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             1880..1906
                     /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             1913..1939
                     /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             1955..2917
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1955..2917
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2933..2986
                     /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             2993..3700
                     /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(3138..3156)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3406..3425
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(3817..3835)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3842..3976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3866..3885)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3920..3939
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(4068..4205,4206..4217)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4275..4292)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4275..4291)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4284..4306)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4433..4888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4520..4539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4730..4751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4914..5018
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5019..5087,5088..5697,1..182)
                     /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(5237..5256)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
       61 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      181 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      241 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      301 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      361 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      421 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      481 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
      541 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      601 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      661 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      721 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      781 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      841 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      901 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      961 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1021 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1081 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     1141 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1201 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1261 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1321 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     1381 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatctgctg
     1441 ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa gtacttgagt aattttactt
     1501 gattactgta cttaagtatt atttttgggg atttttactt tacttgagta caattaaaaa
     1561 tcaatacttt tacttttact taattacatt tttttagaaa aaaaagtact ttttactcct
     1621 tacaatttta tttacagtca aaaagtactt attttttgga gatcacttgg gcccggctcg
     1681 agctacgtac ttaagcttga ggatcccatc gattcgaatt ctcggtacct agtcgacctt
     1741 cgcgaaggcc tatttaggtg acactataga agagtacaat tcaaggttaa gcggccgcaa
     1801 gctagattgg cattccggta ctgttggtaa agccaccatg gccacctatg acgtcccaga
     1861 ttacgcaagt ttgcccgggt atgatgttcc tgattatgct agcctcccgg gttacgatgt
     1921 gcccgactat gcctcacttc caggcagatc tcctatgaag gataacaccg tgccactgaa
     1981 attgattgcc ctgttagcga acggtgaatt tcactctggc gagcagttgg gtgaaacgct
     2041 gggaatgagc cgggcggcta ttaataaaca cattcagaca ctgcgtgact ggggcgttga
     2101 tgtctttacc gttccgggta aaggatacag cctgcctgag cctatccagt tacttaatgc
     2161 taaacagata ttgggtcagc tggatggcgg tagtgtagcc gtgctgccag tgattgactc
     2221 cacgaatcag taccttcttg atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc
     2281 agaataccag caggctggcc gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc
     2341 aaacttatat ttgtcgatgt tctggcgtct ggaacaaggc ccggcggcgg cgattggttt
     2401 aagtctggtt atcggtatcg tgatggcgga agtattacgc aagctgggtg cagataaagt
     2461 tcgtgttaaa tggcctaatg acctctatct gcaggatcgc aagctggcag gcattctggt
     2521 ggagctgact ggcaaaactg gcgatgcggc gcaaatagtc attggagccg ggatcaacat
     2581 ggcaatgcgc cgtgttgaag agagtgtcgt taatcagggg tggatcacgc tgcaggaagc
     2641 ggggatcaat ctcgatcgta atacgttggc ggccatgcta atacgtgaat tacgtgctgc
     2701 gttggaactc ttcgaacaag aaggattggc accttatctg tcgcgctggg aaaagctgga
     2761 taattttatt aatcgcccag tgaaacttat cattggtgat aaagaaatat ttggcatttc
     2821 acgcggaata gacaaacagg gggctttatt acttgagcag gatggaataa taaaaccctg
     2881 gatgggcggt gaaatatccc tgcgtagtgc agaaaaagat atcggatcag gagagggcag
     2941 gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc gggcccagat ctatggtgag
     3001 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     3061 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     3121 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     3181 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     3241 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     3301 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     3361 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     3421 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     3481 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     3541 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     3601 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     3661 gggccgccac tccaccggcg gcatggacga gctgtacaag ggaggaggaa gatctaagct
     3721 gaaccctcct gatgagagtg gccccggctg catgagctgc aagtgtgtgc tctcctgatc
     3781 tagaactata gtgagtcgta ttacgtatcc ggactcccta tagtgagtcg tattaaccgg
     3841 tgatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3901 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3961 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     4021 ggtgtgggag gttttttgat atcccgggtt taaacgcatg cagatctaat actcaagtac
     4081 aattttaatg gagtactttt ttacttttac tcaagtaaga ttctagccag atacttttac
     4141 ttttaattga gtaaaatttt ccctaagtac ttgtactttc acttgagtaa aatttttgag
     4201 tactttttac acctctgtca agaaccatat gccggtaccc aatcgcccta taggacgtcg
     4261 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4321 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4381 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     4441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4621 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4741 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4861 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     4921 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4981 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5041 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5101 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5161 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5221 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5281 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5341 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5401 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5461 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5521 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5581 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5641 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggct
//
