LOCUS       sequence-128095-        4230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing CMV for generation of GMAP-compatible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4230)
  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 4230)
  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 CMV
FEATURES             Location/Qualifiers
     source          1..4230
                     /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"
     enhancer        365..744
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        745..948
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     899..919
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(1117..1136)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1118..1136)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1143..1160)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1143..1159)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1152..1174)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1297..1599
                     /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     1478..1497
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1948..2742
                     /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             1948..2742
                     /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(2002..2021)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2612..2631
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2943..2952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2949..3323
                     /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      3461..4049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3950..3969
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4203..4220
                     /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 gttttttgcg ctgcttcgcg atgtacgggc cagatatacg
      361 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      421 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      481 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      541 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      601 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      661 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      721 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      781 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      841 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      901 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      961 agagaaccca ctgcttactg gcttatcgaa attgacccga cattagcgct acagcttaag
     1021 cgggatccac tagtaacggc cgccagtgtg ctggaattct gcagatatcc atcacactgg
     1081 cggccgctcg agcatgcatc tagagggccc aattcgccct atagtgagtc gtattacaat
     1141 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     1201 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     1261 cgcccttccc aacagttgcg cagcctatac gtacggcagt ttaaggttta cacctataaa
     1321 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgccgggg
     1381 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1441 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1501 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1561 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggcat gagattatca
     1621 aaaaggatct tcacctagat ccttttcacg tagaaagcca gtccgcagaa acggtgctga
     1681 ccccggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga
     1741 aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca
     1801 gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa
     1861 gtaaactgga tggctttctc gccgccaagg atctgatggc gcaggggatc aagctctgat
     1921 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     1981 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2041 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2101 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     2161 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     2221 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     2281 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     2341 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     2401 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     2461 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     2521 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     2581 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     2641 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     2701 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaattattaa cgcttacaat
     2761 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacaggtg
     2821 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2881 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatag cacgtgagga
     2941 gggccaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg
     3001 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact
     3061 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg
     3121 tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt
     3181 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
     3241 gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact
     3301 tcgtggccga ggagcaggac tgacacgtgc taaaacttca tttttaattt aaaaggatct
     3361 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3421 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3481 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3541 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3601 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3661 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3721 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3781 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3841 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3901 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3961 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4021 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4081 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4141 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4201 gcagcgagtc agtgagcgag gaagcggaag
//
