LOCUS       sequence-334820-        6483 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 6483)
  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 6483)
  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-50%GC-40bp
FEATURES             Location/Qualifiers
     source          1..6483
                     /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             1491..2681
                     /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(1843..1862)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(3035..3054)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3036..3054)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3061..3078)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3061..3077)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3070..3092)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3215..3517
                     /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     3396..3415
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3866..4660
                     /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             3866..4660
                     /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(3920..3939)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4530..4549
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4860..4869
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4866..5240
                     /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      5378..5966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5867..5886
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6120..6137
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6254..6275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6290..6295,6296..6313,6314..6320)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6328..6344
                     /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     6333..6355
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6352..6368
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6352..6368
                     /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 gcatttgtct
     1321 agatcgactg acgtacgagt caacgtctgg atccattttt atggatccag acgttgactc
     1381 gtacgtcagt cgatctagac cccgctgtct agaggatccg actatcgacc tcagcgcttt
     1441 aatgcggtag tttatcacag ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta
     1501 acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc ataggcttgg
     1561 ttatgccggt actgccgggc ctcttgcggg atatcgtcca ttccgacagc atcgccagtc
     1621 actatggcgt gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg
     1681 gagcactgtc cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta cttggagcca
     1741 ctatcgacta cgcgatcatg gcgaccacac ccgtcctgtg gatcctctac gccggacgca
     1801 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca
     1861 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta
     1921 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc
     1981 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     2041 cgcataaggg agagcgtcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     2101 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     2161 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     2221 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     2281 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     2341 tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct
     2401 tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc
     2461 tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta
     2521 ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga
     2581 gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg
     2641 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     2701 cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa
     2761 tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac
     2821 gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc
     2881 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     2941 acgcgagcga acgtgaagcg actgcaaggg cgaattctgc agatatccat cacactggcg
     3001 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt attacaattc
     3061 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3121 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3181 cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca cctataaaag
     3241 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgccggggcg
     3301 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     3361 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     3421 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     3481 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa
     3541 aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc
     3601 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
     3661 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
     3721 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt
     3781 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca
     3841 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3901 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3961 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4021 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     4081 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4141 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4201 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4261 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4321 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4381 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4441 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4501 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4561 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4621 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt
     4681 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
     4741 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4801 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgaggaggg
     4861 ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     4921 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     4981 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     5041 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     5101 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     5161 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     5221 tggccgagga gcaggactga cacgtgctaa aacttcattt ttaatttaaa aggatctagg
     5281 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5341 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5401 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5461 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5521 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5581 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5641 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5701 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5761 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5821 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5881 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5941 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6001 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6061 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6121 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     6181 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     6241 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     6301 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     6361 gctatgacca tgattacgcc aagctattta ggtgacgcgt tagaatactc aagctatgca
     6421 tcaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct ggaattcgcc
     6481 ctt
//
