LOCUS       sequence-330267-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of the dead variant (D106A) of HaloTag7 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  AUTHORS   Wilhelm J, Kuhn S, Tarnawski M, Gotthard G, Tunnermann J, Tanzer T, 
            Karpenko J, Mertes N, Xue L, Uhrig U, Reinstein J, Hiblot J, 
            Johnsson K
  TITLE     Kinetic and Structural Characterization of the Self-Labeling Protein
            Tags HaloTag7, SNAP-tag, and CLIP-tag.
  JOURNAL   Biochemistry. 2021 Aug 24;60(33):2560-2575. doi: 
            10.1021/acs.biochem.1c00258. Epub 2021 Aug 2.
  PUBMED    34339177
REFERENCE   2  (bases 1 to 5992)
  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"; doi: 
            "10.1021/acs.biochem.1c00258"; journalName: "Biochemistry"; date: 
            "2021-08-24- 24"; volume: "60"; issue: "33"; pages: "2560-2575"
COMMENT     Sequence Label: pET51b-His-TEV-HaloTag7_dead_mt
FEATURES             Location/Qualifiers
     source          1..5992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(582..1373,1374..1442))
                     /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     1205..1224
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1443..1535)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1616..2204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2105..2124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2358..2375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2390..2529
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2455..2474)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2615..2637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2631..2822)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3394..4476)
                     /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     4438..4457
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4477..4554)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4760..4779
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4867..4886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4867..4885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4886..4910
                     /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             4925..4947
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4957..4986
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4987..5007
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5906..5924)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5920..5967
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
      481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2401 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2461 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2521 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2581 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2641 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2701 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2761 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2821 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2881 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2941 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3001 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3061 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3121 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3181 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3241 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     3301 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3361 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3421 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3481 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3541 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3601 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3661 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3721 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3781 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3841 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3901 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3961 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4021 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4081 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4141 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4201 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4261 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4321 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4381 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4441 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4501 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4561 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     4621 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     4681 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     4741 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     4801 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     4861 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     4921 taattttgtt taactttaag aaggagatat accatgcatc accaccatca tcaccatcac
     4981 caccacgaaa acctgtattt tcagggcatc ggtactggct ttccattcga cccccattat
     5041 gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct
     5101 gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat
     5161 gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa
     5221 ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc
     5281 ctgggtctgg aagaggtcgt cctggtcatt cacgcctggg gctccgctct gggtttccac
     5341 tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct
     5401 atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc
     5461 accgacgtcg gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg
     5521 atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg
     5581 aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag
     5641 ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc
     5701 ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc
     5761 ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg
     5821 caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc tactctggag
     5881 atttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5941 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at
//
