LOCUS       sequence-128118-        4283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing mCherry for generation of GMAP-compatible 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4283)
  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 4283)
  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 mCherry
FEATURES             Location/Qualifiers
     source          1..4283
                     /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..1045
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MASKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYKS"
     primer_bind     745..764
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1170..1189)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1171..1189)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1196..1213)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1196..1212)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1205..1227)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1350..1652
                     /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     1531..1550
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2001..2795
                     /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             2001..2795
                     /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(2055..2074)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2665..2684
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2996..3005
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3002..3376
                     /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      3514..4102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4003..4022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4256..4273
                     /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 catggcaagc aagggcgagg aggataacat
      361 ggccatcatc aaggaattta tgcgcttcaa agttcacatg gagggctccg tgaacggcca
      421 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc aaacagccaa
      481 gctgaaggtt accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcaatt
      541 tatgtatggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
      601 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
      661 gaccgtgacc caggactcct ccctgcaaga cggcgagttc atctacaaag ttaagctgcg
      721 gggaaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
      781 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
      841 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
      901 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
      961 cgaggactac accatcgtgg aacaatacga acgcgccgag ggccgccact ccaccggcgg
     1021 catggacgag ctgtacaagt cttgaaaacg ttgttgtttg gggttgaatt actctggatc
     1081 cactagtaac ggccgccagt gtgctggaat tctgcagata tccatcacac tggcggccgc
     1141 tcgagcatgc atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg
     1201 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     1261 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     1321 cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga
     1381 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga
     1441 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     1501 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     1561 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     1621 acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga
     1681 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     1741 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
     1801 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
     1861 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     1921 ggatggcttt ctcgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga
     1981 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     2041 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     2101 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     2161 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     2221 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     2281 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     2341 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     2401 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     2461 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     2521 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     2581 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     2641 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     2701 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     2761 tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga
     2821 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacag gtggcacttt
     2881 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2941 tccgctcatg agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac
     3001 catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
     3061 cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg
     3121 tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga
     3181 caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga
     3241 ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca
     3301 gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
     3361 cgaggagcag gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa
     3421 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3481 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3541 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3601 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3661 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3721 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3781 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3841 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3901 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3961 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4021 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4081 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4141 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4201 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4261 gtcagtgagc gaggaagcgg aag
//
