LOCUS       sequence-370413-        7110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  chromosomal integration downstream of glmS gene via Tn7 system of a 
            cassette containing IPTG-inducible GFPmut3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7110)
  AUTHORS   Avraham, Roi
  TITLE     Bacterial fluorescent reporters and tags toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7110)
  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: 
            "Unpublished"
COMMENT     Sequence Label: R6K_tac_GFPmut3_ChInt
FEATURES             Location/Qualifiers
     source          1..7110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     mobile_element  181..346
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        join(487..492,493..508,509..515)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    523..539
                     /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     528..550
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             609..1322
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(639..667)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1262..1282
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        1392..1469
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1470..2552
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1489..1508)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    complement(2887..2934)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        join(3011..3016,3017..3033,3034..3039)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3150..3944
                     /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"
     primer_bind     complement(3204..3223)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3814..3833
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3996..4043)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      complement(4094..4180)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(4283..4377)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4405..4421)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4406..4422)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4836..4855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4955..4977
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5015..5033)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5101..5205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5206..5274,5275..6066)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5424..5443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6367..6755
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            join(7024..7110,1..22)
                     /note="incP origin of transfer"
ORIGIN
        1 tcctgctctg cgaggctggc cgataagctc tgataccgct cgccgcagcc gaacgaccga
       61 gcgcagcggg tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
      121 cgcgcgttgg ccgattcatt aatgcagcgc ccaaacataa caggaagaaa aatgccccgc
      181 tgtgggcgga caaaatagtt gggaactggg aggggtggaa atggagtttt taaggattat
      241 ttagggaaga gtgacaaaat agatgggaac tgggtgtagc gtcgtaagct aatacgaaaa
      301 ttaaaaatga caaaatagtt tggaactaga tttcacttat ctggttcttg aggcggttaa
      361 aagagccgta ctcttctccg atgtcgacta ggccatgatg ctcattctgt gggaccaaaa
      421 cgaaaaaaca ccctttcggg tgtcttttct ggaatttggt actggcaaat attctgaaat
      481 gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat
      541 ttcacacagg aaacagaatt cgagctcggt acccggggat cctctagatt taagaaggag
      601 atatacatat gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
      661 tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa
      721 catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc
      781 caacacttgt cactactttc ggttatggtg ttcaatgctt tgcgagatac ccagatcata
      841 tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta
      901 tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
      961 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg
     1021 gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa
     1081 agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac
     1141 tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca
     1201 accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
     1261 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca
     1321 aataaatgtc cagacctgca gccaagcttc gacaattcgc gcgcgaaggc gaagcggcat
     1381 gcatttacgt tgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc
     1441 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1501 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1561 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1621 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1681 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1741 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1801 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1861 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1921 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1981 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     2041 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     2101 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     2161 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     2221 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     2281 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2341 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2401 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2461 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2521 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagcgc
     2581 gaattgtcga agcttggctg ttttggcgga tgagttcatg tggctgaccg ttctgttgtc
     2641 tctcgctctt ccgagtagac gaacaataag gcctccctaa cggggggcct tttttattga
     2701 taacaaaagt cagtgcttcc gctatttcca aaataccggg ctaatacggt ttaaacgacc
     2761 tcctggattt gctcagacag ccttttcgtc attcgtttca gccaaaaaac ttaagaccgc
     2821 cggtcttgtc cactaccttg cagtaatgcg gtggacagga tcggcggttt tcttttctct
     2881 tctcaagaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcctcctg
     2941 aacggccata agaacgaagg ctgtctgttg aactctcgag ccgtggaaac ggatgaaggc
     3001 acgaacccag ttgacataag cctgttcggt tcgtaaactg taatgcaagt agcgtatgcg
     3061 ctcaaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg
     3121 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3181 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3241 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3301 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3361 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3421 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3481 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3541 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3601 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3661 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3721 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3781 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3841 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3901 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3961 cgaaatgact cgagcatcag cttcaaaagc gctctgaagt tcctatactt tctagagaat
     4021 aggaacttcg gaataggtac ttcaagatcc ccaattcgag ctcatcgtcc gggccgcaag
     4081 ctcctagcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
     4141 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctcaag ctagagagtc
     4201 attaccccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc
     4261 cactcatcgc agtctagctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg
     4321 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     4381 gctcagctaa ttaagctagc ttatcgatac cgtcgacctc gaaccccacg cccctcttta
     4441 atacgacggg caatttgcac ttcagaaaat gaagagtttg ctttagccat aacaaaagtc
     4501 cagtatgctt tttcacagca taactggact gatttcagtt tacaactatt ctgtctagtt
     4561 taagacttta ttgtcatagt ttagatctat tttgttcagt ttaagacttt attgtccgcc
     4621 cacacctgct tacgcagggc atccatttat tactcaaccg taaccgattt tgccaggtta
     4681 cgcggctggt cctctagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4741 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     4801 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4861 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4921 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4981 tcaccgtcgt caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5041 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5101 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5161 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5221 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5281 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5341 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5401 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5461 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5521 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5581 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5641 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5701 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5761 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5821 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5881 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtcccgcgg tatcattgca
     5941 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6001 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6061 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6121 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg cagttattgg
     6241 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6301 tgatttaaaa cttcattttt aatttttgcg gccgcaagat ccggccacga tgcgtccggc
     6361 gtagaggatc tgaagatcag cagttcaacc tgttgatagt acgtactaag ctctcatgtt
     6421 tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga actaaaccct
     6481 catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt tcacgtacta
     6541 agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag cctctaaggt
     6601 tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagctttta aggtttaacg
     6661 gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc tctcaaagca
     6721 attttgagtg acacaggaac acttaacggc tgacatggga attagccatg gcatcacagt
     6781 atcgtgatga cagaggcagg gagtgcggcc tttttacggt tcctggcctt ttgctggcct
     6841 tttgctcaca tgttctttcc tgcgttatca ggggattcct taaggtatac tttccgctgc
     6901 ataaccctgc ttcggggtca ttatagcgat tttttcggta tatccatcct ttttcgcacg
     6961 atatacagga ttttgccaaa gggttcgtgt agactttcct tggtgtatcc aacggcgtca
     7021 gccgggcagg ataggtgaag taggcccacc cgcgagcggg tgttccttct tcactgtccc
     7081 ttattcgcac ctggcggtgc tcaacgggaa
//
