LOCUS       Exported                6274 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid encoding the bacterial expression of NbALFA for ALFA-tag as 
            an EGFP-fusion.
ACCESSION   .
VERSION     .
KEYWORDS    pET51b(+)_EGFP_NbALFA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6274)
  AUTHORS   Farrants H, Tarnawski M, Muller TG, Otsuka S, Hiblot J, Koch B, 
            Kueblbeck M, Krausslich HG, Ellenberg J, Johnsson K
  TITLE     Chemogenetic Control of Nanobodies.
  JOURNAL   Nat Methods. 2020 Feb 17. pii: 10.1038/s41592-020-0746-7. doi: 
            10.1038/s41592-020-0746-7.
  PUBMED    32066961
REFERENCE   2  (bases 1 to 6274)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6274
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             97..120
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             127..141
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             151..867
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             151..867
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(196..217)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(457..476)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     804..825
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1270..1299
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1322..1340)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1336..1383
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1420..1875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1507..1526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1717..1738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1990..2850)
                     /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     2613..2632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2851..2943)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3024..3612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3513..3532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3766..3783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3798..3937
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3863..3882)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4023..4045
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4039..4230)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4802..5884)
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5846..5865
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5885..5962)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6168..6187
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gcaagctgga gccacccgca gttcgaaaag
      121 ggtgcagatg acgacgacaa ggtaccgcat atggtgagca agggcgagga gctgttcacc
      181 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      241 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      301 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      361 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      421 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      481 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      541 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      601 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
      661 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      721 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
      781 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
      841 actctcggca tggacgagct gtacaagagc ggcgaagtgc agcttcagga gagtggtggt
      901 ggcttggtgc agccaggtgg ctccttacgg ctgtcctgta ccgcatctgg cgttacgatt
      961 agcgcgctga atgcgatggc tatggggtgg tatcgccaag caccgggaga acgtcgtgtg
     1021 atggtagccg ctgtatcgga acgtggcaat gccatgtatc gcgaaagcgt tcaaggccgc
     1081 tttaccgtta cccgcgattt cacgaacaaa atggtcagtc tgcagatgga caacctgaaa
     1141 ccggaggata cagcggtgta ctactgccat gtcctcgaag atcgcgttga tagctttcac
     1201 gactattggg gtcaagggac ccaggtcact gtgtcgtcag gagctccggg cttctcctca
     1261 atttccgctc atcaccacca tcatcaccat caccaccact aattaaccta ggctgctgcc
     1321 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1381 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1441 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1501 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1561 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1621 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1681 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1741 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1801 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1861 taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta
     1921 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1981 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2041 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2101 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     2161 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     2221 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     2281 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     2341 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2401 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2461 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2521 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2581 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2641 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2701 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2761 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2821 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatcat gattgaagca
     2881 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2941 aaataggtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3001 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3061 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3661 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3721 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3781 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3841 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3901 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3961 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4021 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     4081 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     4141 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     4201 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     4261 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4321 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4381 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4441 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4501 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4561 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4621 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4681 gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4741 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4801 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4861 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4921 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4981 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5041 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     5101 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5161 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5221 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5281 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5341 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5401 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5461 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5521 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5581 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5641 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5701 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5761 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5821 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5881 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5941 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6001 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6061 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6121 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6181 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6241 tagaggatcg agatcgatct cgatcccgcg aaat
//
