LOCUS       sequence-334818-        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-66%GC-20bp
FEATURES             Location/Qualifiers
     source          1..6457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      835..1215
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(922..941)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1132..1153
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1456..2646
                     /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(1808..1827)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3000..3019)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3001..3019)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3026..3043)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3026..3042)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3035..3057)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3180..3482
                     /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     3361..3380
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3831..4625
                     /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             3831..4625
                     /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(3885..3904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4495..4514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4825..4834
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4831..5205
                     /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      5343..5931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5832..5851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6085..6102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6219..6240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6255..6260,6261..6278,6279..6285)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6293..6309
                     /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     6298..6320
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6317..6333
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6317..6333
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tcatgtcaat aatcggctgt ctttccttat cattccttct aaatcctcaa atgtattatc
       61 tattgacggc tctaatctat tagttgttag tgctcctaaa gatattttag ataaccttcc
      121 tcaattcctt tcaactgttg atttgccaac tgaccagata ttgattgagg gtttgatatt
      181 tgaggttcag caaggtgatg ctttagattt ttcatttgct gctggctctc agcgtggcac
      241 tgttgcaggc ggtgttaata ctgaccgcct cacctctgtt ttatcttctg ctggtggttc
      301 gttcggtatt tttaatggcg atgttttagg gctatcagtt cgcgcattaa agactaatag
      361 ccattcaaaa atattgtctg tgccacgtat tcttacgctt tcaggtcaga agggttctat
      421 ctctgttggc cagaatgtcc cttttattac tggtcgtgtg actggtgaat ctgccaatgt
      481 aaataatcca tttcagacga ttgagcgtca aaatgtaggt atttccatga gcgtttttcc
      541 tgttgcaatg gctggcggta atattgttct ggatattacc agcaaggccg atagtttgag
      601 ttcttctact caggcaagtg atgttattac taatcaaaga agtattgcta caacggttaa
      661 tttgcgtgat ggacagactc ttttactcgg tggcctcact gattataaaa acacttctca
      721 ggattctggc gtaccgttcc tgtctaaaat ccctttaatc ggcctcctgt ttagctcccg
      781 ctctgattct aacgaggaaa gcacgttata cgtgctcgtc aaagcaacca tagtacgcgc
      841 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      901 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      961 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1021 tacggcacct cgaccccaaa aaacttgatt tgggtgatga ttcacgtagt gggccatcgc
     1081 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1141 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     1201 ttttgccgat ttcggaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     1261 ggaccgcttg ctgcaactct ctcagcgggc acggccaggc tcccgccctc agcatttgct
     1321 gctggacgac gtgctccatt tttatggagc acgtcgtcca gcagctttgc tgtctagagg
     1381 atccgactat cgacctcagc gctttaatgc ggtagtttat cacagttaaa ttgctaacgc
     1441 agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc
     1501 ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc
     1561 gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata tgcgttgatg
     1621 caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg ccgcccagtc
     1681 ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac cacacccgtc
     1741 ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1801 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1861 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1921 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     1981 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat gcccttgaga
     2041 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     2101 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     2161 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     2221 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     2281 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg
     2341 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc
     2401 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag
     2461 cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcattggacc gctgatcgtc
     2521 acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc
     2581 gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg
     2641 acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca
     2701 atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg
     2761 cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac
     2821 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     2881 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc aagggcgaat
     2941 tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg cccaattcgc
     3001 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3061 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3121 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta tacgtacggc
     3181 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
     3241 gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     3301 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     3361 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     3421 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     3481 aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc acgtagaaag
     3541 ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa
     3601 gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc
     3661 tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg
     3721 gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat
     3781 ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3841 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3901 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3961 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     4021 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4081 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4141 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4201 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4261 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4321 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     4381 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4441 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4501 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4561 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4621 tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg
     4681 gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4741 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4801 cttcaataat agcacgtgag gagggccacc atggccaagt tgaccagtgc cgttccggtg
     4861 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     4921 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     4981 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     5041 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     5101 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     5161 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctaaaactt
     5221 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5281 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5341 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5401 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5461 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5521 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5581 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5641 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5701 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5761 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5821 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5881 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5941 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6001 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6061 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6121 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6181 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     6241 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     6301 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct atttaggtga
     6361 cgcgttagaa tactcaagct atgcatcaag cttggtaccg agctcggatc cactagtaac
     6421 ggccgccagt gtgatggata tctgcagaat tcgccct
//
