LOCUS       sequence-128115-        4289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing GFP for generation of GMAP-compatible 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4289)
  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 4289)
  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 GFP
FEATURES             Location/Qualifiers
     source          1..4289
                     /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             join(332..334,335..337,338..1051)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(377..398)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(638..657)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     985..1006
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1176..1195)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1177..1195)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1202..1219)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1202..1218)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1211..1233)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1356..1658
                     /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     1537..1556
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2007..2801
                     /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             2007..2801
                     /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(2061..2080)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2671..2690
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3002..3011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3008..3382
                     /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      3520..4108
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4009..4028
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4262..4279
                     /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 catggtgagc aagggcgagg agctgttcac
      361 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      421 gtctggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      481 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      541 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      601 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
      661 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
      721 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
      781 cgtctatatc atggccgaca agcagaagaa cggcatcaag gcgaacttca agatccgcca
      841 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
      901 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
      961 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1021 cactctcggc atggacgagc tgtacaagta aaaacgttgt tgtttggggt tgaattactc
     1081 tggatccact agtaacggcc gccagtgtgc tggaattctg cagatatcca tcacactggc
     1141 ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg tattacaatt
     1201 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     1261 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     1321 gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa
     1381 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     1441 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     1501 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     1561 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     1621 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa
     1681 aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa cggtgctgac
     1741 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     1801 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     1861 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     1921 taaactggat ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc
     1981 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2041 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2101 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2161 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     2221 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2281 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2341 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2401 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2461 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2521 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2581 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2641 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2701 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     2761 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt
     2821 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg
     2881 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2941 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagc acgtgaggag
     3001 ggccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     3061 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
     3121 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     3181 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     3241 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     3301 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     3361 cgtggccgag gagcaggact gacacgtgct aaaacttcat ttttaattta aaaggatcta
     3421 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3481 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3541 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3601 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3661 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3721 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3781 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3841 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3901 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3961 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4021 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4081 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4141 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4201 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4261 cagcgagtca gtgagcgagg aagcggaag
//
