LOCUS       40924_29771        7292 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Vector for in vivo biotinylation of a fusion protein in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7292)
  AUTHORS   Tonia Rex
  TITLE     Tonia Rex lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7292)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7292)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(104..292)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        788..865
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             866..1945
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1961..1982
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2191..2219
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    2227..2243
                     /label=lac operator
                     /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)."
     CDS             2313..3413
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     CDS             3462..3473
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     CDS             3551..3571
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             3638..3655
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             3664..3687
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      3699..3785
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3877..3904
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        3931..4022
                     /label=AmpR promoter
     CDS             4023..4880
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4971..5559
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5725..5744
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     protein_bind    5847..5871
                     /label=lac operator
                     /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)."
     RBS             5886..5908
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5915..6877
                     /codon_start=1
                     /label=BirA
                     /note="E. coli biotin protein ligase"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             6916..6933
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      7000..7047
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     7177..7194
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
       61 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      121 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac
      181 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
      241 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
      301 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
      361 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
      421 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
      481 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
      541 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
      601 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
      661 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
      721 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
      781 aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
      841 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
      901 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
      961 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1021 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1081 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     1141 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     1201 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     1261 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     1321 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     1381 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     1441 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     1501 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     1561 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     1621 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatattt cggtagtggg
     1681 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     1741 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     1801 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     1861 caatacacaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1921 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     1981 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
     2041 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     2101 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     2161 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     2221 gtggaattgt gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt
     2281 cacgagcaat tgaccaacaa ggaccataga ttatgaaaat cgaagaaggt aaactggtaa
     2341 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
     2401 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
     2461 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
     2521 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
     2581 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
     2641 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
     2701 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
     2761 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
     2821 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
     2881 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
     2941 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
     3001 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
     3061 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaatgccg
     3121 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
     3181 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     3241 aagagttggt gaaagatccg cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
     3301 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     3361 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
     3421 gttctaacaa caacaacaat aacaataaca acaacctcgg gatcgaggga aggatttcac
     3481 atatgtccat gggcggccgc gatatcgtcg acaagaggtg atatcgatcg gagctctcga
     3541 gccaaccact gaggatctgt actttcagag cgataacgat ggatccctag gactgaacga
     3601 catcttcgaa gctcagaaaa tcgaatggca cggatcccat catcaccatc accactagaa
     3661 ttcgattaca aggatgacga cgataagtaa ataagcttca aataaaacga aaggctcagt
     3721 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     3781 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
     3841 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
     3901 tttttgcgtt tctacaaact ctttcggtcc gttgtttatt tttctaaata cattcaaata
     3961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4201 ccgaagaacg tttcccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4261 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4921 atttccttag gactgagcgt caaccccgta gaaaagatca aaggatcttc ttgagatcct
     4981 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5041 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5101 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     5161 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5221 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5281 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5341 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5401 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5461 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5521 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5581 ttacggttcc tggccttttg ctggcctttt gctcacatgt tccggtaccg cgcccaacag
     5641 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5701 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     5761 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     5821 gcgaaattaa tacgactcac tagaggggaa ttgtgagcgg ataacaattc ccctctagaa
     5881 ataattttgt ttaactttaa gaaggagata taccatgaag gataacaccg tgccactgaa
     5941 attgattgcc ctgttagcga acggtgaatt tcactctggc gagcagttgg gtgaaacgct
     6001 gggaatgagc cgggcggcta ttaataaaca cattcagaca ctgcgtgact ggggcgttga
     6061 tgtctttacc gttccgggta aaggatacag cctgcctgag cctatccagt tacttaatgc
     6121 taaacagata ttgggtcagc tggatggcgg tagtgtagcc gtgctgccag tgattgactc
     6181 cacgaatcag taccttcttg atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc
     6241 agaataccag caggctggcc gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc
     6301 aaacttatat ttgtcgatgt tctggcgtct ggaacaaggc ccggcggcgg cgattggttt
     6361 aagtctggtt atcggtatcg tgatggcgga agtattacgc aagctgggtg cagataaagt
     6421 tcgtgttaaa tggcctaatg acctctatct gcaggatcgc aagctggcag gcattctggt
     6481 ggagctgact ggcaaaactg gcgatgcggc gcaaatagtc attggagccg ggatcaacat
     6541 ggcaatgcgc cgtgttgaag agagtgtcgt taatcagggg tggatcacgc tgcaggaagc
     6601 ggggatcaat ctcgatcgta atacgttggc ggccatgcta atacgtgaat tacgtgctgc
     6661 gttggaactc ttcgaacaag aaggattggc accttatctg tcgcgctggg aaaagctgga
     6721 taattttatt aatcgcccag tgaaacttat cattggtgat aaagaaatat ttggcatttc
     6781 acgcggaata gacaaacagg gggctttatt acttgagcag gatggaataa taaaaccctg
     6841 gatgggcggt gaaatatccc tgcgtagtgc agaaaaaggt aagctttgat gaggactagt
     6901 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     6961 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     7021 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atgctagcac
     7081 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7141 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7201 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     7261 taaggtgcac tgtgactggg tcatggctgc gc
//
