LOCUS       sequence-334826-        6463 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 6463)
  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 6463)
  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-75%GC-20bp
FEATURES             Location/Qualifiers
     source          1..6463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(126..142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(126..142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(139..161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    150..166
                     /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)."
     promoter        complement(join(174..180,181..198,199..204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    219..240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(357..374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(528..1116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(608..627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1254..1628)
                     /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"
     regulatory      1625..1634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1834..2628)
                     /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             complement(1834..2628)
                     /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(1945..1964)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2555..2574
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2977..3279)
                     /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     complement(3079..3098)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3402..3424
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3416..3433
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3417..3433
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3440..3459
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3440..3458
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      4362..4742
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4449..4468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4659..4680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4989..6179
                     /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(5341..5360)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aagggcgaat tctgcagata tccatcacac tggcggccgt tactagtgga tccgagctcg
       61 gtaccaagct tgatgcatag cttgagtatt ctaacgcgtc acctaaatag cttggcgtaa
      121 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
      181 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      241 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      301 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
      361 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      421 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      481 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      541 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      601 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      661 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      721 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      781 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      841 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      901 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      961 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1021 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1081 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1141 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1201 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttagcac gtgtcagtcc
     1261 tgctcctcgg ccacgaagtg cacgcagttg ccggccgggt cgcgcagggc gaactcccgc
     1321 ccccacggct gctcgccgat ctcggtcatg gccggcccgg aggcgtcccg gaagttcgtg
     1381 gacacgacct ccgaccactc ggcgtacagc tcgtccaggc cgcgcaccca cacccaggcc
     1441 agggtgttgt ccggcaccac ctggtcctgg accgcgctga tgaacagggt cacgtcgtcc
     1501 cggaccacac cggcgaagtc gtcctccacg aagtcccggg agaacccgag ccggtcggtc
     1561 cagaactcga ccgctccggc gacgtcgcgc gcggtgagca ccggaacggc actggtcaac
     1621 ttggccatgg tggccctcct cacgtgctat tattgaagca tttatcaggg ttattgtctc
     1681 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1741 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa
     1801 ataccgcatc aggaaattgt aagcgttaat aattcagaag aactcgtcaa gaaggcgata
     1861 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     1921 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     1981 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     2041 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct
     2101 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     2161 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     2221 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     2281 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     2341 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     2401 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag
     2461 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     2521 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     2581 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     2641 tcatcctgtc tcttgatcag agcttgatcc cctgcgccat cagatccttg gcggcaagaa
     2701 agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa
     2761 ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact
     2821 gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct
     2881 gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtga aaaggatcta
     2941 ggtgaagatc ctttttgata atctcatgcc tgacatttat attccccaga acatcaggtt
     3001 aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac
     3061 ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg
     3121 caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg
     3181 gatcaccatc cgtcgccccg gcgtgtcaat aatatcactc tgtacatcca caaacagacg
     3241 ataacggctc tctcttttat aggtgtaaac cttaaactgc cgtacgtata ggctgcgcaa
     3301 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     3361 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     3421 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc ctctagatgc
     3481 atgctcgagc ggccgccagt gtgatggata tctgcagaat tcgcccttca tgtcaataat
     3541 cggctgtctt tccttatcat tccttctaaa tcctcaaatg tattatctat tgacggctct
     3601 aatctattag ttgttagtgc tcctaaagat attttagata accttcctca attcctttca
     3661 actgttgatt tgccaactga ccagatattg attgagggtt tgatatttga ggttcagcaa
     3721 ggtgatgctt tagatttttc atttgctgct ggctctcagc gtggcactgt tgcaggcggt
     3781 gttaatactg accgcctcac ctctgtttta tcttctgctg gtggttcgtt cggtattttt
     3841 aatggcgatg ttttagggct atcagttcgc gcattaaaga ctaatagcca ttcaaaaata
     3901 ttgtctgtgc cacgtattct tacgctttca ggtcagaagg gttctatctc tgttggccag
     3961 aatgtccctt ttattactgg tcgtgtgact ggtgaatctg ccaatgtaaa taatccattt
     4021 cagacgattg agcgtcaaaa tgtaggtatt tccatgagcg tttttcctgt tgcaatggct
     4081 ggcggtaata ttgttctgga tattaccagc aaggccgata gtttgagttc ttctactcag
     4141 gcaagtgatg ttattactaa tcaaagaagt attgctacaa cggttaattt gcgtgatgga
     4201 cagactcttt tactcggtgg cctcactgat tataaaaaca cttctcagga ttctggcgta
     4261 ccgttcctgt ctaaaatccc tttaatcggc ctcctgttta gctcccgctc tgattctaac
     4321 gaggaaagca cgttatacgt gctcgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4381 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4441 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4501 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4561 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4621 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4681 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4741 ggaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     4801 caactctctc agcgggcacg gccaggctcc cgccctcagc atttgcctcc gtgggacgga
     4861 gcgtgccatt tttatggcac gctccgtccc acggaggctt tgctgtctag aggatccgac
     4921 tatcgacctc agcgctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg
     4981 caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg
     5041 ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat atcgtccatt
     5101 ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg atgcaatttc
     5161 tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca gtcctgctcg
     5221 cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc gtcctgtgga
     5281 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     5341 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     5401 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     5461 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     5521 gcttcctaat gcaggagtcg cataagggag agcgtcgacc gatgcccttg agagccttca
     5581 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     5641 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     5701 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     5761 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     5821 aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga
     5881 cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc
     5941 ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag
     6001 gatcgctcgc ggctcttacc agcctaactt cgatcattgg accgctgatc gtcacggcga
     6061 tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat
     6121 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa
     6181 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt
     6241 cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc
     6301 catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg
     6361 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     6421 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgc
//
