LOCUS       sequence-272055-        6766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the bacterial expression of LAMA-R59 for ALFA-tag 
            as an EGFP-fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6766)
  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 6766)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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(+)_EGFP-LAMA-R59 (for ALFA-tag)
FEATURES             Location/Qualifiers
     source          1..6766
                     /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             join(151..153,154..156,157..867)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(151..153,154..156,157..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             1762..1791
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1814..1832)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1828..1875
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1912..2367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1999..2018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2209..2230
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2482..3273,3274..3342))
                     /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     3105..3124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3343..3435)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3516..4104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4005..4024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4258..4275
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4290..4429
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4355..4374)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4515..4537
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4531..4722)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5294..6376)
                     /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     6338..6357
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6377..6454)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6660..6679
                     /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 acgtctgcct gccgatctcg cctggtttaa acgcaacacc
     1081 ttaaataaac ccgtgattat gggccgccat acctgggaat caatcggtcg tccgttgcca
     1141 ggacgcaaaa atattatcct cagcagtcaa ccgggtacgg acgatcgcgt aacgtgggtg
     1201 aagtcggtgg atgaagccat cgcggcgtgt ggtgacgtac cagaaatcat ggtgattggc
     1261 ggcggtcgcg tttatgaaca gttcttgcca aaagcgcaaa aactgtatct gacgcatatc
     1321 gacgcagaag tggaaggcga cacccatttc ccggattacg agccggatga ctgggaatcg
     1381 gtattcagcg aattccacga tgctgatgcg cagaactctc acagctattg ctttgagatt
     1441 ctggagcggc ggggcggcgg cggcggcatg atcagtctga ttgcggcgtt agcggtagat
     1501 cgcgttatcg gcatggaaaa cgccatgccg tggaacggca atgccatgta tcgcgaaagc
     1561 gttcaaggcc gctttaccgt tacccgcgat ttcacgaaca aaatggtcag tctgcagatg
     1621 gacaacctga aaccggagga tacagcggtg tactactgcc atgtcctcga agatcgcgtt
     1681 gatagctttc acgactattg gggtcaaggg acccaggtca ctgtgtcgtc aggagctccg
     1741 ggcttctcct caatttccgc tcatcaccac catcatcacc atcaccacca ctaattaacc
     1801 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1861 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1921 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1981 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2041 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2101 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2161 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2221 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2281 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2341 ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag
     2401 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2461 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2521 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2581 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2641 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2701 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2761 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2821 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2881 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2941 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3001 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3061 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3121 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3181 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3241 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3301 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc
     3361 atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3421 agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga
     3481 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3601 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3661 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3721 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3781 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3841 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3901 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3961 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4021 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4081 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4141 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4201 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4261 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4321 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4381 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4441 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4501 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4561 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4621 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4681 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4741 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4801 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4861 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4921 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4981 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     5041 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     5101 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     5161 cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     5221 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     5281 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5341 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     5401 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     5461 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     5521 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     5581 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     5641 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     5701 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     5761 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     5821 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     5881 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     5941 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     6001 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     6061 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     6121 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6181 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     6241 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     6301 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     6361 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6421 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     6481 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     6541 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     6601 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     6661 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     6721 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaat
//
