LOCUS       sequence-334825-        6457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Force-activating hairpin for 3'-to-5' helicase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6457)
  AUTHORS   Taylor AMK, Okoniewski SR, Uyetake L, Perkins TT
  TITLE     Force-Activated DNA Substrates for In Situ Generation of ssDNA and 
            Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay.
  JOURNAL   Methods Mol Biol. 2022;2478:273-312. doi: 
            10.1007/978-1-0716-2229-2_10.
  PUBMED    36063324
REFERENCE   2  (bases 1 to 6457)
  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: "Methods Mol
            Biol."; date: "2022"; volume: "2478:273-312. doi"; pages: " 
            10.1007/978-1-0716-2229-2_10"
COMMENT     Sequence Label: hp-100%GC-20bp
FEATURES             Location/Qualifiers
     source          1..6457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      834..1214
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(921..940)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1131..1152
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1455..2645
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(1807..1826)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2999..3018)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3000..3018)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3025..3042)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3025..3041)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3034..3056)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3179..3481
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3360..3379
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3830..4624
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3830..4624
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3884..3903)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4494..4513
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4824..4833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4830..5204
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      5342..5930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5831..5850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6084..6101
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6218..6239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6254..6259,6260..6277,6278..6284)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6292..6308
                     /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)."
     primer_bind     6297..6319
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6316..6332
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6316..6332
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catgtcaata atcggctgtc tttccttatc attccttcta aatcctcaaa tgtattatct
       61 attgacggct ctaatctatt agttgttagt gctcctaaag atattttaga taaccttcct
      121 caattccttt caactgttga tttgccaact gaccagatat tgattgaggg tttgatattt
      181 gaggttcagc aaggtgatgc tttagatttt tcatttgctg ctggctctca gcgtggcact
      241 gttgcaggcg gtgttaatac tgaccgcctc acctctgttt tatcttctgc tggtggttcg
      301 ttcggtattt ttaatggcga tgttttaggg ctatcagttc gcgcattaaa gactaatagc
      361 cattcaaaaa tattgtctgt gccacgtatt cttacgcttt caggtcagaa gggttctatc
      421 tctgttggcc agaatgtccc ttttattact ggtcgtgtga ctggtgaatc tgccaatgta
      481 aataatccat ttcagacgat tgagcgtcaa aatgtaggta tttccatgag cgtttttcct
      541 gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga tagtttgagt
      601 tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgctac aacggttaat
      661 ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa cacttctcag
      721 gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt tagctcccgc
      781 tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat agtacgcgcc
      841 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      901 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      961 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1021 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     1081 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1141 gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat
     1201 tttgccgatt tcggaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     1261 gaccgcttgc tgcaactctc tcagcgggca cggccaggct cccgccctca gcatttgccg
     1321 cgggccggcg cgcgccattt ttatggcgcg cgccggcccg cggctttgct gtctagagga
     1381 tccgactatc gacctcagcg ctttaatgcg gtagtttatc acagttaaat tgctaacgca
     1441 gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt catcctcggc accgtcaccc
     1501 tggatgctgt aggcataggc ttggttatgc cggtactgcc gggcctcttg cgggatatcg
     1561 tccattccga cagcatcgcc agtcactatg gcgtgctgct agcgctatat gcgttgatgc
     1621 aatttctatg cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc
     1681 tgctcgcttc gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc
     1741 tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1801 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1861 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     1921 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1981 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgaccgatg cccttgagag
     2041 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2101 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     2161 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     2221 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     2281 agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt
     2341 tcgcgacgcg aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg
     2401 ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc
     2461 ttcaaggatc gctcgcggct cttaccagcc taacttcgat cattggaccg ctgatcgtca
     2521 cggcgattta tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg
     2581 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     2641 cctgaatgga agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa
     2701 tcaattcttg cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc
     2761 gtccgccatc tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg
     2821 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     2881 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgca agggcgaatt
     2941 ctgcagatat ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc
     3001 ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3061 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3121 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca
     3181 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     3241 tgatattatt gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     3301 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     3361 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     3421 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     3481 aatgtcaggc atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc
     3541 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
     3601 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct
     3661 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg
     3721 taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg
     3781 gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3841 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3901 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3961 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4021 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4081 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4141 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4201 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4261 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4321 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4381 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4441 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4501 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4561 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4621 ctgaattatt aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg
     4681 tatttcacac cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4741 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4801 ttcaataata gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc
     4861 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     4921 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     4981 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     5041 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     5101 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     5161 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc
     5221 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5281 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5341 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5401 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5461 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5521 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5581 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5641 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5701 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5761 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5821 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5881 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5941 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6001 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6061 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6121 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6181 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6241 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6301 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcta tttaggtgac
     6361 gcgttagaat actcaagcta tgcatcaagc ttggtaccga gctcggatcc actagtaacg
     6421 gccgccagtg tgatggatat ctgcagaatt cgccctt
//
