LOCUS       sequence-417183-        6158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HaloTag-Spy0128-(Protein of Interest)-Spy0128-SpyCatcher-8xHisTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6158)
  AUTHORS   Tapia-Rojo R, Mora M, Garcia-Manyes S
  TITLE     Single-molecule magnetic tweezers to probe the equilibrium dynamics 
            of individual proteins at physiologically relevant forces and 
            timescales.
  JOURNAL   Nat Protoc. 2024 Mar 11. doi: 10.1038/s41596-024-00965-5.
  PUBMED    38467905
REFERENCE   2  (bases 1 to 6158)
  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 Protoc.
            2024 Mar 11. doi: 10.1038/s41596-024-00965-5."
COMMENT     Sequence Label: pFN18A-HaloTag-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..6158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             70..960
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             973..993
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2800..3138
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     regulatory      2923..2932
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3145..3168
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3262..3280)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3276..3323
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3552..3656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3657..3725,3726..4517)
                     /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     complement(3875..3894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4675..5263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4755..4774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5722..5739)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5892..5978
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6070..6097
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tattttaact ttactaagga
       61 gaattcatca tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc
      121 ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc
      181 ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg
      241 acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg
      301 ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg
      361 gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag
      421 cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc
      481 tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc
      541 ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc
      601 gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt
      661 gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac
      721 atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg
      781 ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa
      841 agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac
      901 aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga gatttccggc
      961 gagccaacca ctgaggatct gtactttcag agcgataacg cgatcgccgg atccgctaca
     1021 acagttcacg gggagactgt tgtaaacgga gccaaactaa cagttacaaa aaaccttgat
     1081 ttagttaata gcaatgcatt aattccaaat acagatttta catttaaaat cgaacctgat
     1141 actactgtca acgaagacgg aaataagttt aaaggtgtag ctttgaacac accgatgact
     1201 aaagtcactt acaccaattc agataaaggt ggatcaaata cgaaaactgc agaatttgat
     1261 ttttcagaag ttacttttga aaaaccaggt gtttattatt acaaagtaac tgaggagaag
     1321 atagataaag ttcctggtgt ttcttatgat acaacatctt acactgttca agttcatgtc
     1381 ttgtggaatg aagagcaaca aaaaccagta gctacttata ttgttggtta taaagaaggt
     1441 agtaaggtgc caattcagtt caaaaatagc ttagattcta ctacattaac ggtgaagaaa
     1501 aaagtttcag gtactggtgg agatcgctct aaagatttta attttggtct gactttaaaa
     1561 gcaaatcagt attataaggc gtcagaaaaa gtcatgattg agaagacaac taaaggtggt
     1621 caagctcctg ttcaaacaga ggctagtata gatcaactct atcattttac cttgaaagat
     1681 ggtgaatcaa tcaaagtcac aaatcttcca gtaggtgtgg attatgttgt cactgaagac
     1741 gattacaaat cagaaaaata tacaaccaac gtggaagtta gtcctcaaga tggagctgta
     1801 aaaaatatcg caggtaattc aactgaacaa gagacatcta ctgataaaga tatgaccatt
     1861 acttttacaa ataaaaaaga ctttgaatcc ggaggaggcg ggggtgctag cgctacaaca
     1921 gttcacgggg agactgttgt aaacggagcc aaactaacag ttacaaaaaa ccttgattta
     1981 gttaatagca atgcattaat tccaaataca gattttacat ttaaaatcga acctgatact
     2041 actgtcaacg aagacggaaa taagtttaaa ggtgtagctt tgaacacacc gatgactaaa
     2101 gtcacttaca ccaattcaga taaaggtgga tcaaatacga aaactgcaga atttgatttt
     2161 tcagaagtta cttttgaaaa accaggtgtt tattattaca aagtaactga ggagaagata
     2221 gataaagttc ctggtgtttc ttatgataca acatcttaca ctgttcaagt tcatgtcttg
     2281 tggaatgaag agcaacaaaa accagtagct acttatattg ttggttataa agaaggtagt
     2341 aaggtgccaa ttcagttcaa aaatagctta gattctacta cattaacggt gaagaaaaaa
     2401 gtttcaggta ctggtggaga tcgctctaaa gattttaatt ttggtctgac tttaaaagca
     2461 aatcagtatt ataaggcgtc agaaaaagtc atgattgaga agacaactaa aggtggtcaa
     2521 gctcctgttc aaacagaggc tagtatagat caactctatc attttacctt gaaagatggt
     2581 gaatcaatca aagtcacaaa tcttccagta ggtgtggatt atgttgtcac tgaagacgat
     2641 tacaaatcag aaaaatatac aaccaacgtg gaagttagtc ctcaagatgg agctgtaaaa
     2701 aatatcgcag gtaattcaac tgaacaagag acatctactg ataaagatat gaccattact
     2761 tttacaaata aaaaagactt tgaaagatcc ggcgccatgg tggacaccct gagtggcctg
     2821 agcagcgagc agggtcagag tggcgacatg accatcgaag aagatagcgc cacccacatc
     2881 aagttcagca agcgcgacga ggacggcaag gaactggcag gcgccaccat ggagctgcgc
     2941 gatagcagcg gcaagaccat cagcacatgg atcagcgacg gccaggtgaa ggacttctac
     3001 ctgtacccgg gcaagtacac cttcgtggaa accgccgccc cggatggcta tgaggttgcc
     3061 accgccatca ccttcaccgt gaacgagcag ggccaggtga ccgtgaacgg caaggccacc
     3121 aagggcgacg cccacatcgg ttcccaccat caccatcacc atcaccatta gggtaccaga
     3181 tcctctagag tcgacctgca ggcatgcaag ctgatccggc tgctaacaaa gcccgaaagg
     3241 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3301 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggttcg cttgctgtcc
     3361 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct
     3421 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc
     3481 accgtttctg cggactggct ttctacgtaa tggtttctta gacgtcaggt ggcacttttc
     3541 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3601 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3661 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3721 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3781 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3841 aacgttttcc aatgatgagc actttcaaag ttctgctatg tggcgcggta ttatcccgta
     3901 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3961 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4021 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca actatcggag
     4081 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4141 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4201 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4261 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4321 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4381 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4441 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4501 tgattaagca ttggtaattc gaaatgaccg accaagcgac gcccaaccgg tatcagctca
     4561 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4621 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4681 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4741 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4801 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4861 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4921 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4981 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5041 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5101 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5161 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5221 tgtttgcaag cagcagatta cgcgcagaaa aaaaggattt caagaagatc ctttgatctt
     5281 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5341 attatcaaaa aggatcttca cctagatcct tttatagtct ggaaatacag gaacgcacgc
     5401 tggatggccc ttcgctggga tggtgaaacc atgaaaaatg gcagcttcag tggattaagt
     5461 gggggtaatg tggcctgtac cctctggttg cataggtatt catacggtta aaatttatca
     5521 ggcgcgattg cggcagtttt tcgggtggtt tgttgccatt tttacctgtc tgctgccgtg
     5581 atcgcgctga acgcgtttta gcggtgcgta caattaaggg attatggtaa atccacttac
     5641 tgtctgccct cgtagccatc gagataaacc gcagtactcc ggccacgatg cgtccggcgt
     5701 agaggatcga gatcttttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     5761 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     5821 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     5881 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     5941 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     6001 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     6061 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt
     6121 ttatttttct aaatacattc aaatatgtat ccgctcat
//
