LOCUS       sequence-375053-        4475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expresses human talin R8 domain flanked between a HaloTag 
            and a SpyTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4475)
  AUTHORS   Dahal N, Sharma S, Phan B, Eis A, Popa I
  TITLE     Mechanical regulation of talin through binding and history-dependent
            unfolding.
  JOURNAL   Sci Adv. 2022 Jul 15;8(28):eabl7719. doi: 10.1126/sciadv.abl7719. 
            Epub 2022 Jul 15.
  PUBMED    35857491
REFERENCE   2  (bases 1 to 4475)
  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: "Sci Adv."; 
            date: "2022-07-15"; volume: "8(28):eabl7719. doi"; pages: " 
            10.1126/sciadv.abl7719. Epub 2022 Jul 15"
COMMENT     Sequence Label: pFN-HaloTag-TalinR8-SpyTag
FEATURES             Location/Qualifiers
     source          1..4475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(112..129)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      282..368
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      460..487
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     569..588
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        569..587
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             618..1508
                     /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             1521..1541
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1959..1997
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             2013..2030
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2127..2145)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2141..2188
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2417..2521
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2522..2590,2591..3382)
                     /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(2740..2759)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3540..4128)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3620..3639)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(4244..4475,1..52)
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 caattaaggg attatggtaa atccacttac tgtctgccct cgtagccatc gagataaacc
       61 gcagtactcc ggccacgatg cgtccggcgt agaggatcga gatcttttca gcctgataca
      121 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
      181 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
      241 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
      301 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
      361 ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg
      421 caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg
      481 gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatgg atctcgatcc cgcgaaatta atacgactca ctatagggta ttttaacttt
      601 actaaggaga attcatcatg gcagaaatcg gtactggctt tccattcgac ccccattatg
      661 tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg
      721 tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg
      781 ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac
      841 cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc
      901 tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact
      961 gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta
     1021 tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc ttccgcacca
     1081 ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt acgctgccga
     1141 tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga
     1201 atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc
     1261 cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc
     1321 cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc
     1381 tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc
     1441 aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg acgctcgaga
     1501 tttccggcga gccaaccact gaggatctgt actttcagag cgataacgcg atcgccggat
     1561 ccgccggtca acagggtctg gttgaaccga cccagtttgc ccgcgccaat caggccatcc
     1621 agatggcatg ccagagctta ggtgagccgg gttgcaccca ggcccaggtt ctgagcgccg
     1681 caaccattgt tgccaaacat accagcgccc tgtgtaatag ctgccgcctg gcaagcgccc
     1741 gtaccacaaa tccgaccgcc aaacgccagt tcgtgcagag cgccaaagaa gtggccaata
     1801 gcacagccaa cctggttaaa accatcaagg ccctggacgg tgcatttacc gaggaaaatc
     1861 gcgcacagtg tcgcgcagca acagccccgc tgctggaagc agttgacaac ctgagcgcct
     1921 tcgccagcaa tcctgagttt agcagcattc cgagatccgc ccacatcgtg atggtggacg
     1981 cctacaagcc gacgaagggg ggttgtagat cccaccatca ccatcaccat tgctaaggta
     2041 ccagatcctc tagagtcgac ctgcaggcat gcaagctgat ccggctgcta acaaagcccg
     2101 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2161 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gttcgcttgc
     2221 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt
     2281 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg
     2341 tcagcaccgt ttctgcggac tggctttcta cgtaatggtt tcttagacgt caggtggcac
     2401 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2461 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2521 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2581 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2641 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2701 cgaagaacgt tttccaatga tgagcacttt caaagttctg ctatgtggcg cggtattatc
     2761 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2821 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2881 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaactat
     2941 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3001 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3061 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3121 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3181 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3241 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3301 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3361 ctcactgatt aagcattggt aattcgaaat gaccgaccaa gcgacgccca accggtatca
     3421 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3481 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3541 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3601 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3661 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3721 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3781 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3841 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3901 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3961 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4021 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4081 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga agatcctttg
     4141 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4201 atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa tacaggaacg
     4261 cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc ttcagtggat
     4321 taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac ggttaaaatt
     4381 tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac ctgtctgctg
     4441 ccgtgatcgc gctgaacgcg ttttagcggt gcgta
//
