LOCUS       sequence-371124-        7348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  chromosomal integration downstream of glmS gene via Tn7 system of a 
            cassette containing IPTG-inducible mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  AUTHORS   Avraham, Roi
  TITLE     Bacterial fluorescent reporters and tags toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7348)
  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            "Unpublished"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_mCherry_ChInt
FEATURES             Location/Qualifiers
     source          1..7348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      40..67
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     89..109
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        219..296
                     /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             297..1379
                     /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(316..335)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    complement(1714..1761)
                     /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(1838..1843,1844..1860,1861..1866)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1977..2771
                     /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(2031..2050)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2641..2660
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(2823..2870)
                     /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(2921..3007)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(3110..3204)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3232..3248)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3233..3249)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3663..3682)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3782..3804
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3842..3860)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3928..4032
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4033..4101,4102..4893)
                     /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(4251..4270)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5194..5582
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            5851..5959
                     /note="incP origin of transfer"
     mobile_element  6118..6283
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        join(6424..6429,6430..6445,6446..6452)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6460..6476
                     /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     6465..6487
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             6499..7209
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6644..6662)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6912..6931
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      7210..7296
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg
       61 gccttttgaa ttcaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga
      121 ttacacatgg catggatgaa ctatacaaat aaatgtccag acctgcagcc aagcttcgac
      181 aattcgcgcg cgaaggcgaa gcggcatgca tttacgttga caccatcgaa tggtgcaaaa
      241 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
      301 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
      361 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
      421 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
      481 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
      541 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
      601 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
      661 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
      721 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
      781 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
      841 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
      901 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
      961 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     1021 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     1081 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     1141 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     1201 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     1261 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     1321 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     1381 cgcaacgcaa ttaatgtaag ttagcgcgaa ttgtcgaagc ttggctgttt tggcggatga
     1441 gttcatgtgg ctgaccgttc tgttgtctct cgctcttccg agtagacgaa caataaggcc
     1501 tccctaacgg ggggcctttt ttattgataa caaaagtcag tgcttccgct atttccaaaa
     1561 taccgggcta atacggttta aacgacctcc tggatttgct cagacagcct tttcgtcatt
     1621 cgtttcagcc aaaaaactta agaccgccgg tcttgtccac taccttgcag taatgcggtg
     1681 gacaggatcg gcggttttct tttctcttct caagaagttc ctatactttc tagagaatag
     1741 gaacttcgga ataggaactt cctcctgaac ggccataaga acgaaggctg tctgttgaac
     1801 tctcgagccg tggaaacgga tgaaggcacg aacccagttg acataagcct gttcggttcg
     1861 taaactgtaa tgcaagtagc gtatgcgctc aaaactggat ggctttcttg ccgccaagga
     1921 tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga
     1981 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2041 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2101 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     2161 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2221 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2281 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     2341 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     2401 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     2461 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     2521 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     2581 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     2641 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     2701 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     2761 agttcttctg agcgggactc tggggttcga aatgactcga gcatcagctt caaaagcgct
     2821 ctgaagttcc tatactttct agagaatagg aacttcggaa taggtacttc aagatcccca
     2881 attcgagctc atcgtccggg ccgcaagctc ctagcggcgg atttgtccta ctcaggagag
     2941 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
     3001 ttatttgatg cctcaagcta gagagtcatt accccaggcg tttaagggca ccaataactg
     3061 ccttaaaaaa attacgcccc gccctgccac tcatcgcagt ctagcttgga ttctcaccaa
     3121 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca
     3181 ttactggatc tatcaacagg agtccaagct cagctaatta agctagctta tcgataccgt
     3241 cgacctcgaa ccccacgccc ctctttaata cgacgggcaa tttgcacttc agaaaatgaa
     3301 gagtttgctt tagccataac aaaagtccag tatgcttttt cacagcataa ctggactgat
     3361 ttcagtttac aactattctg tctagtttaa gactttattg tcatagttta gatctatttt
     3421 gttcagttta agactttatt gtccgcccac acctgcttac gcagggcatc catttattac
     3481 tcaaccgtaa ccgattttgc caggttacgc ggctggtcct ctagctggcg taatagcgaa
     3541 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     3601 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     3661 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3721 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3781 tccgggagct gcatgtgtca gaggttttca ccgtcgtcac cgaaacgcgc gagacgaaag
     3841 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3901 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3961 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4021 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4081 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4141 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4201 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4261 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4321 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4381 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4441 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4501 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4561 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4621 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4681 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4741 gagcgtgggt cccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4801 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4861 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4921 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4981 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5041 gtagaaaaga tcaaaggcag ttattggtgc ctcactgatt aagcattggt aactgtcaga
     5101 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttttgcggcc
     5161 gcaagatccg gccacgatgc gtccggcgta gaggatctga agatcagcag ttcaacctgt
     5221 tgatagtacg tactaagctc tcatgtttca cgtactaagc tctcatgttt aacgtactaa
     5281 gctctcatgt ttaacgaact aaaccctcat ggctaacgta ctaagctctc atggctaacg
     5341 tactaagctc tcatgtttca cgtactaagc tctcatgttt gaacaataaa attaatataa
     5401 atcagcaact taaatagcct ctaaggtttt aagttttata agaaaaaaaa gaatatataa
     5461 ggcttttaaa gcttttaagg tttaacggtt gtggacaaca agccagggat gtaacgcact
     5521 gagaagccct tagagcctct caaagcaatt ttgagtgaca caggaacact taacggctga
     5581 catgggaatt agccatggca tcacagtatc gtgatgacag aggcagggag tgcggccttt
     5641 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcaggg
     5701 gattccttaa ggtatacttt ccgctgcata accctgcttc ggggtcatta tagcgatttt
     5761 ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga
     5821 ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc
     5881 gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc
     5941 tgctctgcga ggctggccga taagctctga taccgctcgc cgcagccgaa cgaccgagcg
     6001 cagcgggtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6061 gcgttggccg attcattaat gcagcgccca aacataacag gaagaaaaat gccccgctgt
     6121 gggcggacaa aatagttggg aactgggagg ggtggaaatg gagtttttaa ggattattta
     6181 gggaagagtg acaaaataga tgggaactgg gtgtagcgtc gtaagctaat acgaaaatta
     6241 aaaatgacaa aatagtttgg aactagattt cacttatctg gttcttgagg cggttaaaag
     6301 agccgtactc ttctccgatg tcgactaggc catgatgctc attctgtggg accaaaacga
     6361 aaaaacaccc tttcgggtgt cttttctgga atttggtact ggcaaatatt ctgaaatgag
     6421 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
     6481 acacaggaaa cagaattcat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
     6541 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     6601 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     6661 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     6721 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     6781 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     6841 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     6901 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     6961 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     7021 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     7081 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     7141 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     7201 tacaagtaac aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     7261 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     7321 gcgaagcaac ggcccggagg gtggcggg
//
