LOCUS       sequence-128083-        4223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing mKusabira-Orange2 for generation of 
            GMAP-compatible gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4223)
  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 4223)
  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 mKO2
FEATURES             Location/Qualifiers
     source          1..4223
                     /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..985
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(1110..1129)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1111..1129)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1136..1153)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1136..1152)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1145..1167)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1290..1592
                     /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     1471..1490
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1941..2735
                     /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             1941..2735
                     /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(1995..2014)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2605..2624
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2936..2945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2942..3316
                     /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      3454..4042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3943..3962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4196..4213
                     /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 catggtgtcc gtgatcaagc ccgagatgaa
      361 gatgcgttac tacatggacg gcagcgtgaa cggccacgag ttcaccatcg agggcgaggg
      421 cacaggcaga ccttacgagg gccaccagga aatgaccctg agagtgacca tggccgaggg
      481 cggacctatg cccttcgcct tcgatctggt gtcccacgtg ttctgctacg gccacagagt
      541 gttcaccaag taccccgagg aaatccccga ctacttcaag caggccttcc ccgagggcct
      601 gagctgggag agaagcctgg aattcgagga cggcggcagc gcctctgtgt ctgcccacat
      661 ctctctgaga ggcaacacct tctaccacaa gagcaagttc accggcgtga acttccccgc
      721 cgacggcccc atcatgcaga accagtctgt ggactgggag cccagcaccg agaagatcac
      781 agcctctgac ggcgtgctga agggcgacgt gaccatgtac ctgaagctgg aaggcggcgg
      841 aaaccacaag tgccagatga agaccaccta caaggccgcc aaagaaatcc tggaaatgcc
      901 cggcgaccac tacatcggcc acaggctcgt gcgcaagacc gagggcaaca tcaccgaaca
      961 ggtggaagat gccgtggccc acagcaaacg ttgttgtttg gggttgaatt actctggatc
     1021 cactagtaac ggccgccagt gtgctggaat tctgcagata tccatcacac tggcggccgc
     1081 tcgagcatgc atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg
     1141 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     1201 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     1261 cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga
     1321 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga
     1381 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     1441 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     1501 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     1561 acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga
     1621 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     1681 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
     1741 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
     1801 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     1861 ggatggcttt ctcgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga
     1921 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     1981 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     2041 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     2101 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     2161 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     2221 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     2281 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     2341 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     2401 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     2461 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     2521 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     2581 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     2641 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     2701 tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga
     2761 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacag gtggcacttt
     2821 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2881 tccgctcatg agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac
     2941 catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
     3001 cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg
     3061 tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga
     3121 caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga
     3181 ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca
     3241 gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
     3301 cgaggagcag gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa
     3361 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3421 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3481 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3541 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3601 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3661 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3721 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3781 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3841 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3901 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3961 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4021 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4081 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4141 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4201 gtcagtgagc gaggaagcgg aag
//
