LOCUS       sequence-114742-        5429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CFP tagging via transposon insertion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5429)
  AUTHORS   Sheridan DL, Hughes TE
  TITLE     A faster way to make GFP-based biosensors: two new transposons for 
            creating multicolored libraries of fluorescent fusion proteins.
  JOURNAL   BMC Biotechnol. 2004 Aug 18;4:17.
  PUBMED    15317651
REFERENCE   2  (bases 1 to 5429)
  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: "BMC 
            Biotechnol."; date: "2004-08-18"; volume: "4"; pages: "17"
COMMENT     Sequence Label: pBNJ26.3
FEATURES             Location/Qualifiers
     source          1..5429
                     /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"
     misc_feature    337..355
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(449..1243)
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(1508..2218)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(1550..1571)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1899..1918
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2158..2179
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    2228..2246
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     primer_bind     complement(2316..2335)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2317..2335)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2342..2359)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2342..2358)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2351..2373)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2496..2798
                     /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     2677..2696
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3147..3941
                     /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             3147..3941
                     /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"
     regulatory      4142..4151
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4148..4522
                     /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      4660..5248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5149..5168
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5402..5419
                     /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 ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttctgt ctcttataca catctgcccg
      361 ggctcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg
      421 tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg
      481 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
      541 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
      601 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
      661 tcggcaagca ggcatcgcca tgtgtcacga cgagatcctc gccgtcgggc atgcgcgcct
      721 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
      781 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
      841 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
      901 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
      961 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     1021 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
     1081 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     1141 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     1201 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     1261 ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca
     1321 tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg
     1381 gttcgcttgc tgtccataaa accgcccagt ctagctaaag ggcgaattct gcagatatcc
     1441 atcacactgg cggccgccac tgtgctggat atctgcagaa ttcggcttta ggcccgggcg
     1501 cgccgccctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
     1561 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca
     1621 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     1681 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag tgggccttga
     1741 tgccgttctt ctgcttgtcg gcggtgatat agacgttgtg gctgatgtag ttgtactcca
     1801 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     1861 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     1921 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
     1981 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccccaggtc agggtggtca
     2041 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     2101 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     2161 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctcg
     2221 gcgcgccaga tgtgtataag agacagaagg gcgaattctg cagatatcca tcacactggc
     2281 ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg tattacaatt
     2341 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2401 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2461 gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa
     2521 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     2581 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     2641 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     2701 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     2761 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa
     2821 aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa cggtgctgac
     2881 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     2941 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     3001 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     3061 taaactggat ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc
     3121 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3181 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3241 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3301 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3361 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3421 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3481 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3541 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3601 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3661 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3721 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3781 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3841 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3901 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt
     3961 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg
     4021 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4081 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagc acgtgaggag
     4141 ggccaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     4201 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
     4261 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     4321 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     4381 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     4441 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     4501 cgtggccgag gagcaggact gacacgtgct aaaacttcat ttttaattta aaaggatcta
     4561 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4621 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4681 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4741 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4801 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4861 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4921 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4981 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5041 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5101 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5161 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5221 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5281 gggcttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5341 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5401 cagcgagtca gtgagcgagg aagcggaag
//
