LOCUS       Exported                6616 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Donor cassette containing HA-tagged biotin ligase (BirA), a Thosea 
            asigna virus peptide (2A), mCherry protein and SV40 polyA tail, 
            followed by FRT site-flanked Kanamycin selection gene.
ACCESSION   .
VERSION     .
KEYWORDS    pGEM-cytoBirA-2A-mCherry-SV40pA-FKF
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  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 6616)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6616
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(1..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     582..604
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     596..613
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     597..613
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      669..678
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      673..682
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             805..1767
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1783..1836
                     /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"
     primer_bind     2256..2275
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2605..2726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2616..2635)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2670..2689
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2850..2897)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        2991..3095
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3120..3140)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3327..4121
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3381..3400)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3991..4010
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4128..4176
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(4201..4248)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        complement(4377..4395)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4378..4395)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4413..4429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4413..4429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4426..4448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4437..4453
                     /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(4461..4491)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4506..4527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4644..4661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4815..5403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4895..4914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5574..6434)
                     /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     6197..6216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6435..6539)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
      481 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      541 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      601 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc cgacgtcgca
      661 tgctcccggc cgccatggcg gccgcggatg gccacctatg acgtcccaga ttacgcaagt
      721 ttgcccggat atgatgttcc tgattatgct agcctcccgg gttacgatgt gcccgactat
      781 gcctcacttc caggcagatc tcctatgaag gataacaccg tgccactgaa attgattgcc
      841 ctgttagcga acggtgaatt tcactctggc gagcagttgg gtgaaacgct gggaatgagc
      901 cgggcggcta ttaataaaca cattcagaca ctgcgtgact ggggcgttga tgtctttacc
      961 gttccgggaa aaggatacag cctgcctgag cctatccagt tacttaatgc taaacagata
     1021 ttgggtcagc tggatggcgg tagtgtagcc gtgctgccag tgattgactc cacgaatcag
     1081 taccttcttg atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc agaataccag
     1141 caggctggcc gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc aaacttatat
     1201 ttgtcgatgt tctggcgtct ggaacaaggc ccggcggcgg cgattggttt aagtctggtt
     1261 atcggtatcg tgatggcgga agtattacgc aagctgggtg cagataaagt tcgtgttaaa
     1321 tggcctaatg acctctatct gcaggatcgc aagctggcag gcattctggt ggagctgact
     1381 ggcaaaactg gcgatgcggc gcaaatagtc attggagccg ggatcaacat ggcaatgcgc
     1441 cgtgttgaag agagtgtcgt taatcagggg tggatcacgc tgcaggaagc ggggatcaat
     1501 ctcgatcgta atacgttggc ggccatgcta atacgtgaat tacgtgctgc gttggaactc
     1561 ttcgaacaag aaggattggc accttatctg tcgcgctggg aaaagctgga taattttatt
     1621 aatcgcccag tgaaacttat cattggtgat aaagaaatat ttggcatttc acgcggaata
     1681 gacaaacagg gggctttatt acttgagcag gatggaataa taaaaccctg gatgggcggt
     1741 gaaatatccc tgcgtagtgc agaaaaagat atcggatcag gagagggcag gggaagtctt
     1801 ctaacatgcg gggacgtgga ggaaaatccc gggcccggaa gtatggtgtc gaagggcgag
     1861 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1921 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1981 cagaccgcca agcttaaagt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     2041 tcccctcaat tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     2101 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     2161 ggcggcgtgg tgaccgtgac ccaggactcc tccctccaag atggcgagtt catttataaa
     2221 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     2281 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     2341 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     2401 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     2461 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     2521 tccaccggcg gcatggacga gctgtacaag taaagcggcc gcgactctag agtcggggcg
     2581 gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     2641 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2701 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcggg
     2761 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     2821 tcgataagga tccgtcgacc gacctgcttg aagttcctat actttctaga gaataggaac
     2881 ttcggaatag gaacttcaag atccccctgg cgaaaggggg atgtgctgca aggcgattaa
     2941 gttgggtaac gccagggttt tcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3001 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3061 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3121 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3181 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3241 cagaagtatg caaagcatgc atctcaatta gtcagtaacc atagtccgca aagatcgatc
     3301 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3361 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3421 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3481 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3541 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3601 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3661 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3721 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3781 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3841 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3901 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3961 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4021 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4081 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aggggatcgg caataaaaag
     4141 acagaataaa acgcacgggt gttgggtcgt ttgttcggat ccgagcttca aaagcgctct
     4201 gaagttccta tactttctag agaataggaa cttcggaata gtaacttctc catggtagcc
     4261 tccaaaaaag cctcctcact acttctggaa tagctcaatc actagtgaat tcgcggccgc
     4321 ctgcaggtcg accatatggg agagctccca acgcgttgga tgcatagctt gagtattcta
     4381 tagtgtcacc taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4441 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4501 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4561 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4621 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4681 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4741 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4801 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4861 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4921 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4981 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5041 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5101 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5161 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5221 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5281 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5341 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5401 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5461 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5521 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5581 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5641 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5701 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5761 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5821 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5881 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5941 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6001 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6061 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6121 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6181 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6241 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6301 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6361 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6421 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6481 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6541 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
     6601 aaaataccgc atcagg
//
