LOCUS       sequence-128088-        4169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing PuroR for generation of GMAP-compatible 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4169)
  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 4169)
  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 PuroR
FEATURES             Location/Qualifiers
     source          1..4169
                     /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"
     CDS             332..931
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(332..351)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     828..848
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1056..1075)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1057..1075)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1082..1099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1082..1098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1091..1113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1236..1538
                     /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     1417..1436
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1887..2681
                     /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             1887..2681
                     /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(1941..1960)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2551..2570
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2882..2891
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2888..3262
                     /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      3400..3988
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3889..3908
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4142..4159
                     /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 ctcggatcct
      301 aactcgaacg ctagctgtgc gatcgtttac catgaccgag tacaagccca cggtgcgcct
      361 cgccacccgc gatgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
      421 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
      481 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
      541 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
      601 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
      661 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
      721 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
      781 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
      841 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
      901 ctggtgcatg acccgcaagc ccggtgcctg aaaacgttgt tgtttggggt tgaattactc
      961 tggatccact agtaacggcc gccagtgtgc tggaattctg cagatatcca tcacactggc
     1021 ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg tattacaatt
     1081 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     1141 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     1201 gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa
     1261 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     1321 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     1381 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     1441 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     1501 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa
     1561 aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa cggtgctgac
     1621 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     1681 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     1741 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     1801 taaactggat ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc
     1861 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     1921 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     1981 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2041 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     2101 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2161 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2221 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2281 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2341 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2401 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2461 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2521 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2581 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     2641 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt
     2701 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg
     2761 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2821 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagc acgtgaggag
     2881 ggccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     2941 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
     3001 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     3061 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     3121 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     3181 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     3241 cgtggccgag gagcaggact gacacgtgct aaaacttcat ttttaattta aaaggatcta
     3301 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3361 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3421 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3481 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3541 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3601 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3661 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3721 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3781 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3841 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3901 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3961 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4021 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4081 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4141 cagcgagtca gtgagcgagg aagcggaag
//
