LOCUS       sequence-128100-        3655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing HRE for generation of GMAP-compatible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3655)
  AUTHORS   Akama-Garren EH, Joshi NS, Tammela T, Chang GP, Wagner BL, Lee DY, 
            Rideout Iii WM, Papagiannakopoulos T, Xue W, Jacks T
  TITLE     A Modular Assembly Platform for Rapid Generation of DNA Constructs.
  JOURNAL   Sci Rep. 2016 Feb 18;6:16836. doi: 10.1038/srep16836.
  PUBMED    26887506
REFERENCE   2  (bases 1 to 3655)
  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: "Sci Rep."; 
            date: "2016-02-18"; volume: "6:16836. doi"; pages: " 
            10.1038/srep16836"
COMMENT     Sequence Label: TOPO HRE
FEATURES             Location/Qualifiers
     source          1..3655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(542..561)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(543..561)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(568..585)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(568..584)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(577..599)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             722..1024
                     /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     903..922
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1373..2167
                     /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             1373..2167
                     /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(1427..1446)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2037..2056
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2368..2377
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2374..2748
                     /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      2886..3474
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3375..3394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3628..3645
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcag
      301 tgtgagggag tgtaaagctg gtttaggtac cgagctctgt cacgtcctgc acgactctag
      361 ttgtcacgtc ctgcacgact ctagttgtca cgtcctgcac gacgctagct cgagatccga
      421 cccgacatta gcgctacagc ttaagcggga tccactagta acggccgcca gtgtgctgga
      481 attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc
      541 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
      601 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
      661 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg
      721 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
      781 gagtgatatt attgacacgc cggggcgacg gatggtgatc cccctggcca gtgcacgtct
      841 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
      901 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
      961 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     1021 ataaatgtca ggcatgagat tatcaaaaag gatcttcacc tagatccttt tcacgtagaa
     1081 agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac
     1141 aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata
     1201 gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc
     1261 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     1321 atggcgcagg ggatcaagct ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     1381 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     1441 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     1501 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     1561 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     1621 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     1681 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     1741 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     1801 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     1861 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     1921 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     1981 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     2041 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     2101 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     2161 cttctgaatt attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     2221 cggtatttca caccgcatac aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2281 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2341 atgcttcaat aatagcacgt gaggagggcc accatggcca agttgaccag tgccgttccg
     2401 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
     2461 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
     2521 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
     2581 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
     2641 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
     2701 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa
     2761 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2821 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2881 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2941 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3001 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3061 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3121 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3181 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3241 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3301 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3361 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3421 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3481 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3541 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3601 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
