LOCUS       sequence-334824-        5228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  50-0-50%GC-Right-half-120bp-hairpin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5228)
  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 5228)
  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-50-0-50%GC-Right-half-120bp
FEATURES             Location/Qualifiers
     source          1..5228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             226..1416
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALTQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(578..597)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1826..1842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1826..1842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1839..1861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1850..1866
                     /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(1874..1880,1881..1898,1899..1904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1919..1940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2057..2074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2228..2816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2308..2327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2954..3328)
                     /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      3325..3334
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3534..4328)
                     /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(3534..4328)
                     /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(3645..3664)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4255..4274
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(4677..4979)
                     /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(4779..4798)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     5102..5124
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5116..5133
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5117..5133
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5140..5159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5140..5158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 taaggatcca tttttatgga tccagacgtt gactcgtacg tcagtcgatc tagacataat
       61 ataatttata atataattta taatataatt tattacttca ctgcttgact gtagctgcat
      121 acctgtgacc agagtcccgc tgtctagagg atccgactat cgacctcagc gctttaatgc
      181 ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat
      241 gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg
      301 ccggtactgc cgggcctctt gcgggatatc gtccattccg acagcatcgc cagtcactat
      361 ggcgtgctgc tagcgctata tgcgttgacg caatttctat gcgcacccgt tctcggagca
      421 ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc
      481 gactacgcga tcatggcgac cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg
      541 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
      601 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
      661 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
      721 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
      781 aagggagagc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
      841 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
      901 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
      961 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     1021 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     1081 gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
     1141 attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc
     1201 aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc
     1261 ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca
     1321 tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg
     1381 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
     1441 acggattcac cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc
     1501 aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc
     1561 gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga
     1621 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     1681 agcgaacgtg aagcgactgc aagggcgaat tctgcagata tccatcacac tggcggccgt
     1741 tactagtgga tccgagctcg gtaccaagct tgatgcatag cttgagtatt ctaacgcgtc
     1801 acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1861 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1921 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1981 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2041 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2101 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2161 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2221 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2281 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2341 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2401 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2461 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2521 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2581 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2641 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2701 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2761 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2821 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2881 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2941 gttttagcac gtgtcagtcc tgctcctcgg ccacgaagtg cacgcagttg ccggccgggt
     3001 cgcgcagggc gaactcccgc ccccacggct gctcgccgat ctcggtcatg gccggcccgg
     3061 aggcgtcccg gaagttcgtg gacacgacct ccgaccactc ggcgtacagc tcgtccaggc
     3121 cgcgcaccca cacccaggcc agggtgttgt ccggcaccac ctggtcctgg accgcgctga
     3181 tgaacagggt cacgtcgtcc cggaccacac cggcgaagtc gtcctccacg aagtcccggg
     3241 agaacccgag ccggtcggtc cagaactcga ccgctccggc gacgtcgcgc gcggtgagca
     3301 ccggaacggc actggtcaac ttggccatgg tggccctcct cacgtgctat tattgaagca
     3361 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3421 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg tgaaataccg
     3481 cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat aattcagaag
     3541 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     3601 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     3661 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     3721 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     3781 tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     3841 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     3901 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     3961 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     4021 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     4081 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     4141 tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     4201 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     4261 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     4321 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag agcttgatcc cctgcgccat
     4381 cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca
     4441 gagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc
     4501 tatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt
     4561 gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct
     4621 ttctacgtga aaaggatcta ggtgaagatc ctttttgata atctcatgcc tgacatttat
     4681 attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca
     4741 gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc
     4801 cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc
     4861 agacgtgcac tggccagggg gatcaccatc cgtcgccccg gcgtgtcaat aatatcactc
     4921 tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc
     4981 cgtacgtata ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     5041 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     5101 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc
     5161 gaattgggcc ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat
     5221 tcgccctt
//
