LOCUS       Exported                6460 bp ds-DNA     circular SYN 03-SEP-2019
DEFINITION  Vector for in vivo biotinylation of a fusion protein in bacteria..
ACCESSION   .
VERSION     .
KEYWORDS    MAK464
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  AUTHORS   Tonia Rex
  TITLE     Tonia Rex lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Feb 27, 2020 from SnapGene 5.0.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6460
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     85..107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="color: ?; sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(101..292)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        788..865
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
                     /note="color: #ffffff; direction: RIGHT"
     CDS             866..1948
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(885..904)
                     /label=LacI-R
                     /note="LacI, reverse primer"
                     /note="color: ?; sequence: GGCATACTCTGCGACATCGT"
     promoter        2191..2219
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
                     /note="This forward directional feature has 3 segments:
                        1: 2191 .. 2196 / #ffffff / -35
                        2: 2197 .. 2212 / #ffffff
                        3: 2213 .. 2219 / #ffffff / -10"
     protein_bind    2227..2243
                     /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)."
                     /note="color: #31849b"
     primer_bind     2232..2254
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
                     /note="color: ?; sequence: AGCGGATAACAATTTCACACAGG"
     primer_bind     2241..2264
                     /label=pMALseqF1
                     /note="color: ?; sequence: caatttcacacaggaaacagccag; added:
                     2019-04-18"
     primer_bind     2277..2305
                     /label=MAK418F
                     /note="color: ?; sequence: ttttcacgagcaattgaccaacaaggacc; 
                     added: 2019-03-08"
     CDS             2313..2396
                     /codon_start=1
                     /gene="malE"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This gene encodes a signal sequence that can direct 
                     MBP to the bacterial periplasm."
                     /note="This feature has 2 segments:
                        1: 2313 .. 2390 / #cc99b2 / signal sequence
                        2: 2391 .. 2396 / #cc99b2
                     Cleavage site after base 2390"
                     /translation="MKIKTGARILALSALTTMMFSASALAKI"
     primer_bind     complement(2377..2412)
                     /label=MAK398invR
                     /note="color: ?; sequence: 
                     CCTGCGCCATggatccgattttggcgagagccgagg"
     primer_bind     2382..2405
                     /label=MAK438F
                     /note="color: ?; sequence: gctctcgccaaaatcggatccATG; added:
                     2019-05-09"
     CDS             2403..2750
                     /codon_start=1
                     /label=GFPnano
                     /note="color: #993366"
                     /translation="MAQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGK
                     EREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGF
                     EYWGQGTQVTVS"
     misc_feature    2484..2513
                     /label=CDR1
                     /note="color: #993366; direction: RIGHT"
     misc_feature    2556..2585
                     /label=CDR2
                     /note="color: #993366; direction: RIGHT"
     misc_feature    2703..2720
                     /label=CDR3
                     /note="color: #993366; direction: RIGHT"
     primer_bind     complement(2731..2768)
                     /label=MAK439R
                     /note="color: ?; sequence: 
                     gtcgttcagtccgatatcGCTAACGGTAACCTGGGTGC; added: 2019-05-09"
     primer_bind     2739..2760
                     /label=MAK464F
                     /note="color: ?; sequence: GTTACCGTTAGCgatatcggac; added: 
                     2019-06-18"
     CDS             2757..2798
                     /codon_start=1
                     /label=AviTAG
                     /note="color: #993366"
                     /translation="GLNDIFEAQKIEWH"
     primer_bind     complement(2775..2841)
                     /label=MAK461R
                     /note="color: ?; sequence: 
                     ccttgtaatcgaattcttaGTGGTGATGATGGTGATGGGAGCCgtgccattcgattttc
                     tgagcttc; added: 2019-06-18"
     CDS             2805..2822
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     CDS             2832..2855
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     terminator      2867..2953
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
     primer_bind     complement(2929..2949)
                     /label=pMALseqR
                     /note="color: ?; sequence: gtcctactcaggagagcgttc"
     terminator      3045..3072
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
     promoter        3100..3190
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             3191..4051
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 3191 .. 3259 / #ccffcc / signal sequence
                        2: 3260 .. 4051 / #ccffcc
                     Cleavage site after base 3259"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3409..3428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: ?; sequence: ATAATACCGCGCCACATAGC"
     rep_origin      4139..4727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     4628..4647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: ?; sequence: GGGAAACGCCTGGTATCTTT"
     primer_bind     4893..4912
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
                     /note="color: ?; sequence: GGTGATGTCGGCGATATAGG"
     protein_bind    5015..5039
                     /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)."
                     /note="color: #31849b"
     RBS             5054..5076
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
     CDS             5083..6045
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /note="color: #993366"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             6084..6101
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     primer_bind     complement(6154..6172)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
                     /note="color: ?; sequence: GCTAGTTATTGCTCAGCGG"
     terminator      6168..6215
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff"
     primer_bind     6345..6362
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: ?; sequence: AGCGAGTCAGTGAGCGAG"
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 aaaaacaggt gcacgcatcc
     2341 tcgcattatc cgcattaacg acgatgatgt tttccgcctc ggctctcgcc aaaatcggat
     2401 ccatggcgca ggttcagctg gttgaaagcg gtggtgcact ggttcagcct ggtggtagcc
     2461 tgcgtctgag ctgtgcagca agcggttttc cggttaatcg ttatagcatg cgttggtatc
     2521 gtcaggcacc gggtaaagaa cgtgaatggg ttgcaggtat gagcagtgcc ggtgatcgta
     2581 gcagctatga agatagcgtt aaaggtcgtt ttaccatcag ccgtgatgat gcacgtaata
     2641 ccgtttatct gcaaatgaat agcctgaaac cggaagatac cgcagtgtat tattgcaatg
     2701 ttaacgtggg ctttgaatat tggggtcagg gcacccaggt taccgttagc gatatcggac
     2761 tgaacgacat cttcgaagct cagaaaatcg aatggcacgg ctcccatcac catcatcacc
     2821 actaagaatt cgattacaag gatgacgacg ataagtaaat aagcttcaaa taaaacgaaa
     2881 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     2941 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     3001 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     3061 ggatggcctt tttgcgtttc tacaaactct ttcggtccgt tgtttatttt tctaaataca
     3121 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3181 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3241 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3301 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3361 ttttcgcccc gaagaacgtt tcccaatgat gagcactttt aaagttctgc tatgtggcgc
     3421 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3481 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3541 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3601 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3661 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3721 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3781 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3841 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3901 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3961 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4021 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4081 ttagattgat ttccttagga ctgagcgtca accccgtaga aaagatcaaa ggatcttctt
     4141 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4201 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4261 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4321 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4381 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4441 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4501 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4561 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4621 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4681 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4741 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc cggtaccgcg
     4801 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     4861 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     4921 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     4981 tcgatcccgc gaaattaata cgactcacta gaggggaatt gtgagcggat aacaattccc
     5041 ctctagaaat aattttgttt aactttaaga aggagatata ccatgaagga taacaccgtg
     5101 ccactgaaat tgattgccct gttagcgaac ggtgaatttc actctggcga gcagttgggt
     5161 gaaacgctgg gaatgagccg ggcggctatt aataaacaca ttcagacact gcgtgactgg
     5221 ggcgttgatg tctttaccgt tccgggtaaa ggatacagcc tgcctgagcc tatccagtta
     5281 cttaatgcta aacagatatt gggtcagctg gatggcggta gtgtagccgt gctgccagtg
     5341 attgactcca cgaatcagta ccttcttgat cgtatcggag agcttaaatc gggcgatgct
     5401 tgcattgcag aataccagca ggctggccgt ggtcgccggg gtcggaaatg gttttcgcct
     5461 tttggcgcaa acttatattt gtcgatgttc tggcgtctgg aacaaggccc ggcggcggcg
     5521 attggtttaa gtctggttat cggtatcgtg atggcggaag tattacgcaa gctgggtgca
     5581 gataaagttc gtgttaaatg gcctaatgac ctctatctgc aggatcgcaa gctggcaggc
     5641 attctggtgg agctgactgg caaaactggc gatgcggcgc aaatagtcat tggagccggg
     5701 atcaacatgg caatgcgccg tgttgaagag agtgtcgtta atcaggggtg gatcacgctg
     5761 caggaagcgg ggatcaatct cgatcgtaat acgttggcgg ccatgctaat acgtgaatta
     5821 cgtgctgcgt tggaactctt cgaacaagaa ggattggcac cttatctgtc gcgctgggaa
     5881 aagctggata attttattaa tcgcccagtg aaacttatca ttggtgataa agaaatattt
     5941 ggcatttcac gcggaataga caaacagggg gctttattac ttgagcagga tggaataata
     6001 aaaccctgga tgggcggtga aatatccctg cgtagtgcag aaaaaggtaa gctttgatga
     6061 ggactagtgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     6121 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     6181 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     6241 gctagcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6301 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6361 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     6421 accgcatata aggtgcactg tgactgggtc atggctgcgc
//
