LOCUS       sequence-128079-        3955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO backbone for GMAP genes with sites #2 and #5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3955)
  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 3955)
  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 2-5
FEATURES             Location/Qualifiers
     source          1..3955
                     /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(381..402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(642..661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(842..861)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(843..861)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(868..885)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(868..884)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(877..899)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1022..1324
                     /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     1203..1222
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1673..2467
                     /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             1673..2467
                     /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(1727..1746)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2337..2356
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2668..2677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2674..3048
                     /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      3186..3774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3675..3694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3928..3945
                     /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 gatcgtttaa acaccatggt gtctaagggc gaagagctgt
      361 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
      421 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
      481 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
      541 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
      601 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
      661 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctggtttaaa
      721 cgttgttgtt tggggttgaa ttactctgga tccactagta acggccgcca gtgtgctgga
      781 attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc
      841 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
      901 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
      961 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tatacgtacg
     1021 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     1081 gagtgatatt attgacacgc cggggcgacg gatggtgatc cccctggcca gtgcacgtct
     1141 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     1201 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     1261 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     1321 ataaatgtca ggcatgagat tatcaaaaag gatcttcacc tagatccttt tcacgtagaa
     1381 agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac
     1441 aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata
     1501 gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc
     1561 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     1621 atggcgcagg ggatcaagct ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     1681 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     1741 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     1801 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     1861 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     1921 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     1981 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2041 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     2101 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     2161 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     2221 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     2281 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     2341 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     2401 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     2461 cttctgaatt attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     2521 cggtatttca caccgcatac aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2581 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2641 atgcttcaat aatagcacgt gaggagggcc accatggcca agttgaccag tgccgttccg
     2701 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
     2761 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
     2821 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
     2881 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
     2941 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
     3001 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa
     3061 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3121 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3181 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3241 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3301 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3361 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3421 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3481 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3541 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3601 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3661 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3721 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3781 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3841 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3901 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
