LOCUS       sequence-152593-        5724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCS for cloning promoter to drive 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 5724)
  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 5724)
  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-NLS-BirA-2A-mCherry_Ras
FEATURES             Location/Qualifiers
     source          1..5724
                     /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..1975
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1982..2944
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1982..2944
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2960..3013
                     /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             3020..3727
                     /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(3165..3183)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3433..3452
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(3844..3862)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3869..4003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3893..3912)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3947..3966
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(4095..4232,4233..4244)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4302..4319)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4302..4318)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4311..4333)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4460..4915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4547..4566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4757..4778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4941..5045
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5046..5114,5115..5724,1..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(5264..5283)
                     /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 tcctccaaaa aagaagagaa aggtacgatc
     1981 tatgaaggat aacaccgtgc cactgaaatt gattgccctg ttagcgaacg gtgaatttca
     2041 ctctggcgag cagttgggtg aaacgctggg aatgagccgg gcggctatta ataaacacat
     2101 tcagacactg cgtgactggg gcgttgatgt ctttaccgtt ccgggtaaag gatacagcct
     2161 gcctgagcct atccagttac ttaatgctaa acagatattg ggtcagctgg atggcggtag
     2221 tgtagccgtg ctgccagtga ttgactccac gaatcagtac cttcttgatc gtatcggaga
     2281 gcttaaatcg ggcgatgctt gcattgcaga ataccagcag gctggccgtg gtcgccgggg
     2341 tcggaaatgg ttttcgcctt ttggcgcaaa cttatatttg tcgatgttct ggcgtctgga
     2401 acaaggcccg gcggcggcga ttggtttaag tctggttatc ggtatcgtga tggcggaagt
     2461 attacgcaag ctgggtgcag ataaagttcg tgttaaatgg cctaatgacc tctatctgca
     2521 ggatcgcaag ctggcaggca ttctggtgga gctgactggc aaaactggcg atgcggcgca
     2581 aatagtcatt ggagccggga tcaacatggc aatgcgccgt gttgaagaga gtgtcgttaa
     2641 tcaggggtgg atcacgctgc aggaagcggg gatcaatctc gatcgtaata cgttggcggc
     2701 catgctaata cgtgaattac gtgctgcgtt ggaactcttc gaacaagaag gattggcacc
     2761 ttatctgtcg cgctgggaaa agctggataa ttttattaat cgcccagtga aacttatcat
     2821 tggtgataaa gaaatatttg gcatttcacg cggaatagac aaacaggggg ctttattact
     2881 tgagcaggat ggaataataa aaccctggat gggcggtgaa atatccctgc gtagtgcaga
     2941 aaaagatatc ggatcaggag agggcagggg aagtcttcta acatgcgggg acgtggagga
     3001 aaatcccggg cccagatcta tggtgagcaa gggcgaggag gataacatgg ccatcatcaa
     3061 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     3121 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     3181 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     3241 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     3301 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     3361 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     3421 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     3481 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     3541 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     3601 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     3661 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     3721 gtacaaggga ggaggaagat ctaagctgaa ccctcctgat gagagtggcc ccggctgcat
     3781 gagctgcaag tgtgtgctct cctgatctag aactatagtg agtcgtatta cgtatccgga
     3841 ctccctatag tgagtcgtat taaccggtga tccagacatg ataagataca ttgatgagtt
     3901 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     3961 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     4021 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttgatatc ccgggtttaa
     4081 acgcatgcag atctaatact caagtacaat tttaatggag tactttttta cttttactca
     4141 agtaagattc tagccagata cttttacttt taattgagta aaattttccc taagtacttg
     4201 tactttcact tgagtaaaat ttttgagtac tttttacacc tctgtcaaga accatatgcc
     4261 ggtacccaat cgccctatag gacgtcgtat tacgcgcgct cactggccgt cgttttacaa
     4321 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4381 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4441 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4501 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4561 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4621 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4681 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4741 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4801 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4861 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     4921 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4981 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5041 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5101 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5161 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5221 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5281 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5341 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5401 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5461 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5521 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5581 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5641 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5701 tgcgctcggc ccttccggct ggct
//
