LOCUS       sequence-450049-        6293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the HUH endonuclease WDV fused at the C termini with 
            Echistatin to target RGD binding integrins with DNA tension sensors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6293)
  AUTHORS   Pawlak MR, Smiley AT, Ramirez MP, Kelly MD, Shamsan GA, Anderson SM,
            Smeester BA, Largaespada DA, Odde DJ, Gordon WR
  TITLE     RAD-TGTs: high-throughput measurement of cellular mechanotype via 
            rupture and delivery of DNA tension probes.
  JOURNAL   Nat Commun. 2023 Apr 28;14(1):2468. doi: 10.1038/s41467-023-38157-6.
  PUBMED    37117218
REFERENCE   2  (bases 1 to 6293)
  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 
            Commun."; date: "2023-04-28"; volume: "14(1):2468. doi"; pages: " 
            10.1038/s41467-023-38157-6"
COMMENT     Sequence Label: WDV-Echistatin
FEATURES             Location/Qualifiers
     source          1..6293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(114..1196)
                     /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     1158..1177
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1197..1274)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1480..1499
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1583..1602
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1583..1601
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1602..1626
                     /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             1641..1663
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1682..1699
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1712..2002
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2607..2624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2677..2695)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2691..2738
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2775..3230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2862..2881)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3072..3093
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3256..3360
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3361..3429,3430..4221)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3579..3598)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4392..4980
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4881..4900
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5134..5151
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5166..5305
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5231..5250)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5391..5413
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5407..5598)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
       61 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
      121 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      181 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
      241 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
      301 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
      361 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
      421 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
      481 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
      541 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
      601 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
      661 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
      721 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
      781 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
      841 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
      901 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
      961 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     1021 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     1081 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     1141 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     1201 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     1261 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     1321 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     1381 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     1441 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     1501 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     1561 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     1621 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgcgtggttc
     1681 tcaccaccac caccaccata tggctagcgg atcggactca gaagtcaatc aagaagctaa
     1741 gccagaggtc aagccagaag tcaagcctga gactcacatc aatttaaagg tgtccgatgg
     1801 atcttcagag atcttcttca agatcaaaaa gaccactcct ttaagaaggc tgatggaagc
     1861 gttcgctaaa agacagggta aggaaatgga ctccttaaga ttcttgtacg acggtattag
     1921 aattcaagct gatcagaccc ctgaagattt ggacatggag gataacgata ttattgaggc
     1981 tcacagagaa cagattggtg gtggatccat ggcaagcagc agcacaccgc gttttcgtgt
     2041 ttatagcaaa tacctgtttc tgacctatcc gcagtgtacc ctggaaccgc agtatgcact
     2101 ggattcactg cgtaccctgc tgaacaaata tgaaccgctg tatattgcag cagttcgtga
     2161 actgcatgaa gatggtagtc cgcatctgca tgttctggtt cagaataaac tgcgtgcaag
     2221 cattaccaat ccgaatgcac tgaatctgcg tatggatacc agtccgttta gcatttttca
     2281 tccgaatatc caggcagcca aagattgtaa tcaggtgcgt gattatatca ccaaagaagt
     2341 tgattccgac gttaataccg cagaatgggg cacctttgtt gcagttagca caccgggtcg
     2401 taaagatcgt gatgccgatc tcgagggtga atgtgaaagc ggtccgtgtt gtcgtaattg
     2461 caaatttctg aaagaaggca ccatttgtaa acgtgcccgt ggtgatgata tggatgatta
     2521 ttgtaatggc aaaacctgtg attgtccgcg taatccgcat aaaggtccgg caaccgctag
     2581 ctaactcgag taataagctt gtcgagcacc accaccacca ccactgagat ccggctgcta
     2641 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2701 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2761 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2821 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2881 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2941 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3001 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3061 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3121 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3181 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3241 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3301 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3361 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3421 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3481 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3541 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3601 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3661 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3721 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3781 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3841 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3901 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3961 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4021 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4081 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4141 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4201 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4261 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4321 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4381 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4441 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4501 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4561 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4621 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4681 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4741 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4801 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4861 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4921 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4981 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5041 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5101 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5161 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     5221 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5281 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5341 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5401 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5461 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5521 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5581 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5641 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5701 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5761 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5821 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5881 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5941 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     6001 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     6061 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     6121 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     6181 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     6241 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cga
//
