LOCUS       sequence-128122-        4357 bp    DNA     circular SYN 14-APR-2025
DEFINITION  TOPO construct expressing mTagBFP2 for generation of GMAP-compatible
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4357)
  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 4357)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 mTagBFP2
FEATURES             Location/Qualifiers
     source          1..4357
                     /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             341..358
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             368..1069
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1244..1263)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1245..1263)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1270..1287)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1270..1286)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1279..1301)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1424..1726
                     /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     1605..1624
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2075..2869
                     /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             2075..2869
                     /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(2129..2148)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2739..2758
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3070..3079
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3076..3450
                     /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      3588..4176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4077..4096
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4330..4347
                     /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 catggggagc caccatcacc atcaccatgg
      361 cagatctatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
      421 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
      481 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
      541 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
      601 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
      661 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
      721 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
      781 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
      841 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata
      901 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
      961 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     1021 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaat gagtatacca
     1081 ccatccacta caagtacatg tgtaatagtc cttgcatgga aacgttgttg tttggggttg
     1141 aattactctg gatccactag taacggccgc cagtgtgctg gaattctgca gatatccatc
     1201 acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta
     1261 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1321 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1381 caccgatcgc ccttcccaac agttgcgcag cctatacgta cggcagttta aggtttacac
     1441 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     1501 gccggggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     1561 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     1621 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     1681 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag
     1741 attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg
     1801 gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc
     1861 aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt
     1921 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc
     1981 ctgcaaagta aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag
     2041 ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     2101 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     2161 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     2221 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     2281 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     2341 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     2401 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     2461 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     2521 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     2581 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     2641 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     2701 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     2761 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     2821 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc
     2881 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2941 acaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3001 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac
     3061 gtgaggaggg ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac
     3121 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag
     3181 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac
     3241 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac
     3301 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc
     3361 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc
     3421 gtgcacttcg tggccgagga gcaggactga cacgtgctaa aacttcattt ttaatttaaa
     3481 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3541 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3601 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3661 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3721 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3781 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3841 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3901 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3961 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4021 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4081 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4141 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4201 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4261 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4321 accgagcgca gcgagtcagt gagcgaggaa gcggaag
//
