LOCUS       sequence-128080-        3945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO backbone for GMAP promoters with sites #1 and #4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3945)
  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 3945)
  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 1-4
FEATURES             Location/Qualifiers
     source          1..3945
                     /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(377..398)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(832..851)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(833..851)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(858..875)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(858..874)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(867..889)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1012..1314
                     /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     1193..1212
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1663..2457
                     /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             1663..2457
                     /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(1717..1736)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2327..2346
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2658..2667
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2664..3038
                     /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      3176..3764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3665..3684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3918..3935
                     /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 gtttaaacac catggtgtct aagggcgaag agctgttcac
      361 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      421 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      481 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      541 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      601 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaaggcccg
      661 cgccgaggtg aagttcgagg gcgagctagc gacccgacat tagcgctaca gcttaagcgg
      721 gatccactag taacggccgc cagtgtgctg gaattcgccc ttaagggcga attctgcaga
      781 tatccatcac actggcggcc gctcgagcct gcatctagag ggcccaattc gccctatagt
      841 gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
      901 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
      961 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag
     1021 gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt
     1081 attgacacgc cggggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat
     1141 aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg
     1201 accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc
     1261 caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca
     1321 ggcatgagat tatcaaaaag gatcttcacc tagatccttt tcacgtagaa agccagtccg
     1381 cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac
     1441 gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg
     1501 gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt
     1561 gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg
     1621 ggatcaagct ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     1681 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     1741 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     1801 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     1861 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     1921 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     1981 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     2041 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     2101 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     2161 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     2221 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     2281 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     2341 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     2401 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt
     2461 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2521 caccgcatac aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2581 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2641 aatagcacgt gaggagggcc accatggcca agttgaccag tgccgttccg gtgctcaccg
     2701 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     2761 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     2821 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     2881 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     2941 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     3001 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa cttcattttt
     3061 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3301 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3841 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3901 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
