LOCUS       sequence-370402-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  chromosomal integration downstream of glmS gene via Tn7 system of a 
            cassette containing IPTG-inducible destabilized GFPmut3 (containing 
            a AAV C-terminal tail).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7552)
  AUTHORS   Avraham, Roi
  TITLE     Bacterial fluorescent reporters and tags toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7552)
  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            "Unpublished"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_dGFPmut3_ChInt
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     mobile_element  134..299
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        join(440..445,446..461,462..468)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    476..492
                     /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     481..503
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             562..1275
                     /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"
     CDS             562..1275
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(592..620)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1215..1235
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        1384..1461
                     /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             1462..2544
                     /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(1481..1500)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     complement(2620..2637)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2790..2876
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2968..2995
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    complement(3282..3329)
                     /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(3406..3411,3412..3428,3429..3434)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3545..4339
                     /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(3599..3618)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4209..4228
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(4391..4438)
                     /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(4489..4575)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(4678..4772)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4800..4816)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4801..4817)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5231..5250)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5350..5372
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5410..5428)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5496..5600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5601..5669,5670..6461)
                     /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(5819..5838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6762..7150
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            7419..7527
                     /note="incP origin of transfer"
ORIGIN
        1 gccgaacgac cgagcgcagc gggtcagtga gcgaggaagc ggaagagcgc ccaatacgca
       61 aaccgcctct ccccgcgcgt tggccgattc attaatgcag cgcccaaaca taacaggaag
      121 aaaaatgccc cgctgtgggc ggacaaaata gttgggaact gggaggggtg gaaatggagt
      181 ttttaaggat tatttaggga agagtgacaa aatagatggg aactgggtgt agcgtcgtaa
      241 gctaatacga aaattaaaaa tgacaaaata gtttggaact agatttcact tatctggttc
      301 ttgaggcggt taaaagagcc gtactcttct ccgatgtcga ctaggccatg atgctcattc
      361 tgtgggacca aaacgaaaaa acaccctttc gggtgtcttt tctggaattt ggtactggca
      421 aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg
      481 agcggataac aatttcacac aggaaacaga attcgagctc ggtacccggg gatcctctag
      541 atttaagaag gagatataca tatgagtaaa ggagaagaac ttttcactgg agttgtccca
      601 attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt
      661 gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta
      721 cctgttccat ggccaacact tgtcactact ttcggttatg gtgttcaatg ctttgcgaga
      781 tacccagatc atatgaaaca gcatgacttt ttcaagagtg ccatgcccga aggttatgta
      841 caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag
      901 tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat
      961 ggaaacattc ttggacacaa attggaatat aactataact cacacaatgt atacatcatg
     1021 gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa cattgaagat
     1081 ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc
     1141 cttttaccag acaaccatta cctgtccaca caatctgccc tttcgaaaga tcccaacgaa
     1201 aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg
     1261 gatgaactat acaaaaggcc tgcagcaaac gacgaaaact acgctgcagc agtttaaatg
     1321 tccagacctg cagccaagct tcgacaattc gcgcgcgaag gcgaagcggc atgcatttac
     1381 gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     1441 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     1501 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     1561 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     1621 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     1681 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     1741 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     1801 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     1861 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     1921 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     1981 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     2041 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     2101 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     2161 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     2221 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     2281 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     2341 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     2401 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     2461 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2521 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc gcgaattgtc
     2581 gaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
     2641 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
     2701 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
     2761 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     2821 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     2881 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     2941 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     3001 ttctacaaat tcatgtggct gaccgttctg ttgtctctcg ctcttccgag tagacgaaca
     3061 ataaggcctc cctaacgggg ggcctttttt attgataaca aaagtcagtg cttccgctat
     3121 ttccaaaata ccgggctaat acggtttaaa cgacctcctg gatttgctca gacagccttt
     3181 tcgtcattcg tttcagccaa aaaacttaag accgccggtc ttgtccacta ccttgcagta
     3241 atgcggtgga caggatcggc ggttttcttt tctcttctca agaagttcct atactttcta
     3301 gagaatagga acttcggaat aggaacttcc tcctgaacgg ccataagaac gaaggctgtc
     3361 tgttgaactc tcgagccgtg gaaacggatg aaggcacgaa cccagttgac ataagcctgt
     3421 tcggttcgta aactgtaatg caagtagcgt atgcgctcaa aactggatgg ctttcttgcc
     3481 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt
     3541 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3601 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3661 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3721 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3781 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3841 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3901 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3961 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4021 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4081 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4141 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4201 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4261 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4321 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgactcgagc atcagcttca
     4381 aaagcgctct gaagttccta tactttctag agaataggaa cttcggaata ggtacttcaa
     4441 gatccccaat tcgagctcat cgtccgggcc gcaagctcct agcggcggat ttgtcctact
     4501 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     4561 ctttcgtttt atttgatgcc tcaagctaga gagtcattac cccaggcgtt taagggcacc
     4621 aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtct agcttggatt
     4681 ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc
     4741 tgaggtcatt actggatcta tcaacaggag tccaagctca gctaattaag ctagcttatc
     4801 gataccgtcg acctcgaacc ccacgcccct ctttaatacg acgggcaatt tgcacttcag
     4861 aaaatgaaga gtttgcttta gccataacaa aagtccagta tgctttttca cagcataact
     4921 ggactgattt cagtttacaa ctattctgtc tagtttaaga ctttattgtc atagtttaga
     4981 tctattttgt tcagtttaag actttattgt ccgcccacac ctgcttacgc agggcatcca
     5041 tttattactc aaccgtaacc gattttgcca ggttacgcgg ctggtcctct agctggcgta
     5101 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5161 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     5221 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5281 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5341 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcgtcaccg aaacgcgcga
     5401 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5461 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5521 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5581 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5641 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5701 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5761 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5821 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5881 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5941 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6001 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6061 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6121 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6181 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6241 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6301 gagccggtga gcgtgggtcc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6361 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6421 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6481 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6541 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6601 cagaccccgt agaaaagatc aaaggcagtt attggtgcct cactgattaa gcattggtaa
     6661 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6721 ttgcggccgc aagatccggc cacgatgcgt ccggcgtaga ggatctgaag atcagcagtt
     6781 caacctgttg atagtacgta ctaagctctc atgtttcacg tactaagctc tcatgtttaa
     6841 cgtactaagc tctcatgttt aacgaactaa accctcatgg ctaacgtact aagctctcat
     6901 ggctaacgta ctaagctctc atgtttcacg tactaagctc tcatgtttga acaataaaat
     6961 taatataaat cagcaactta aatagcctct aaggttttaa gttttataag aaaaaaaaga
     7021 atatataagg cttttaaagc ttttaaggtt taacggttgt ggacaacaag ccagggatgt
     7081 aacgcactga gaagccctta gagcctctca aagcaatttt gagtgacaca ggaacactta
     7141 acggctgaca tgggaattag ccatggcatc acagtatcgt gatgacagag gcagggagtg
     7201 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7261 tatcagggga ttccttaagg tatactttcc gctgcataac cctgcttcgg ggtcattata
     7321 gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt
     7381 cgtgtagact ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc
     7441 ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac
     7501 gggaatcctg ctctgcgagg ctggccgata agctctgata ccgctcgccg ca
//
