LOCUS       sequence-270723-        5782 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid encoding the bacterial expression of ALFA-tag asa SNAP-tag 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5782)
  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 5782)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2020 Feb 17. pii: 10.1038/s41592-020-0746-7. doi: 
            10.1038/s41592-020-0746-7."
COMMENT     Sequence Label: pET51b(+)_ALFA-tag-SNAP
FEATURES             Location/Qualifiers
     source          1..5782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             154..192
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             778..807
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(830..848)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      844..891
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      928..1383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1015..1034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1225..1246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1498..2289,2290..2358))
                     /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     2121..2140
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2359..2451)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2532..3120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3021..3040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3274..3291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3306..3445
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3371..3390)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3531..3553
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3547..3738)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4310..5392)
                     /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     5354..5373
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5393..5470)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5676..5695
                     /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 ccgagccgcc tggaagaaga actgcgccgc
      181 cgcctgaccg aaccgagcgg catggataaa gactgcgaaa tgaaacgcac taccctggac
      241 agcccgctgg gtaaactgga actgagcggt tgcgagcagg gcctgcacga aatcattttc
      301 ctgggcaaag gtacgagcgc tgcggatgcg gtcgaagtac cggctccggc tgcggttctg
      361 ggtggcccgg agccgctgat gcaggctacc gcatggctga acgcatattt tcaccagcca
      421 gaggcgattg aagagttccc tgtaccggcc ctgcatcacc cggtatttca gcaggaatcc
      481 tttacccgtc aggtactgtg gaaactgctg aaagtagtga aattcggcga ggtgatctcc
      541 tattcccatc tggcggcgct ggctggcaac ccagcggcaa ctgcggcggt gaaaaccgct
      601 ctgtctggta atccggtgcc aattctgatc ccgtgccacc gtgtggttca gggcgacctg
      661 gacgtaggtg gctacgaagg cggcctggct gtgaaagaat ggctgctggc gcacgaaggt
      721 caccgtctgg gtaaaccagg cctgggcggt gctccgggct tctcctcaat ttccgctcat
      781 caccaccatc atcaccatca ccaccactaa ttaacctagg ctgctgccac cgctgagcaa
      841 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      901 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      961 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1021 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1081 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1141 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1201 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1261 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1321 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1381 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1441 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1501 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1561 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1621 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     1681 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1741 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1801 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1861 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1921 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1981 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2041 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2101 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2161 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2221 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2281 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2341 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt
     2401 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg
     2461 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2521 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2581 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2641 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     2701 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2761 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2821 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2881 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2941 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3001 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3061 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3121 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3181 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3241 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3301 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3361 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3421 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3481 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3541 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3601 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     3661 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     3721 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     3781 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     3841 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3901 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3961 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4021 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4081 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4141 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt
     4201 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4261 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4321 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4381 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4441 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4501 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4561 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     4621 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4681 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4741 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4801 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4861 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4921 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4981 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5041 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5101 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5161 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5221 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5281 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5341 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5401 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5461 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5521 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5581 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     5641 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     5701 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     5761 atcgatctcg atcccgcgaa at
//
