LOCUS       Exported                4910 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Flag-miniTurbo fusion for proximity labeling.
ACCESSION   .
VERSION     .
KEYWORDS    Flag-miniTurbo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4910)
  TITLE     Expresses Flag-miniTurbo fusion for proximity labeling
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4910)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4910
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1069..1092
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1099..1869
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     promoter        complement(1918..1935)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1940..2074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1964..1983)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2018..2037
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2187..2207)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2187..2205)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2226..2242)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2226..2242)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2239..2261)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2250..2266
                     /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(2274..2304)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2319..2340
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2457..2474)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2628..3216)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2708..2727)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3387..4247)
                     /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     4010..4029
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4248..4352)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4378..4833)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4515..4536)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4727..4746
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat ccaccatgga ttacaaggat
     1081 gacgacgata agatccgcat cccgctgctg aacgctaaac agattctggg acagctggac
     1141 ggcgggagcg tggcagtcct gcctgtggtc gactccacca atcagtacct gctggatcga
     1201 atcggcgagc tgaagagtgg ggatgcttgc attgcagaat atcagcaggc agggagagga
     1261 agcagaggga ggaaatggtt ctctcctttt ggagctaacc tgtacctgag tatgttttgg
     1321 cgcctgaagc ggggaccagc agcaatcggc ctgggcccgg tcatcggaat tgtcatggca
     1381 gaagcgctgc gaaagctggg agcagacaag gtgcgagtca aatggcccaa tgacctgtat
     1441 ctgcaggata gaaagctggc aggcatcctg gtggagctgg ccggaataac aggcgatgct
     1501 gcacagatcg tcattggcgc cgggattaac gtggctatga ggcgcgtgga ggaaagcgtg
     1561 gtcaatcagg gctggatcac actgcaggaa gcagggatta acctggacag gaatactctg
     1621 gccgctatgc tgatccgaga gctgcgggca gccctggaac tgttcgagca ggaaggcctg
     1681 gctccatatc tgtcacggtg ggagaagctg gataacttca tcaatagacc cgtgaagctg
     1741 atcattgggg acaaagagat tttcgggatt agccggggga ttgataaaca gggagccctg
     1801 ctgctggaac aggacggagt tatcaaaccc tggatgggcg gagaaatcag tctgcggtct
     1861 gccgaaaagc tgcagggatc tcagatctcg aattcaaggc ctctcgagcc tctagaacta
     1921 tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt ttggacaaac
     1981 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     2041 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     2101 gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca
     2161 ttgggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     2221 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2281 cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2341 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2401 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2461 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2521 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2581 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2641 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2701 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2761 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2821 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2881 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2941 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3001 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3061 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3121 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3181 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3241 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3301 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3361 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3421 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3481 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3541 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3601 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3661 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3721 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3781 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3841 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3901 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3961 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4021 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4081 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4141 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4201 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4261 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4321 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     4381 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4441 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4501 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4561 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4621 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4681 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4741 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4801 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     4861 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc
//
