LOCUS       sequence-230506-        6422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tn7 tagging vector with a LacI-controlled kill switch 
            counterselection system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   Wiles TJ, Wall ES, Schlomann BH, Hay EA, Parthasarathy R, Guillemin 
            K
  TITLE     Modernized Tools for Streamlined Genetic Manipulation and 
            Comparative Study of Wild and Diverse Proteobacterial Lineages.
  JOURNAL   MBio. 2018 Oct 9;9(5). pii: mBio.01877-18. doi: 
            10.1128/mBio.01877-18.
  PUBMED    30301859
REFERENCE   2  (bases 1 to 6422)
  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: "MBio."; 
            date: "2018-10-9"; volume: "9(5). pii: mBio.01877-18. doi"; pages: "
            10.1128/mBio.01877-18"
COMMENT     Sequence Label: pTn7xKS
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            230..338
                     /note="incP origin of transfer"
     mobile_element  497..662
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     protein_bind    complement(775..822)
                     /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(863..868,869..885,886..891)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1080..1613
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    complement(1743..1790)
                     /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(1836..1922)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(2025..2119)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2147..2163)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2148..2164)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    2553..2574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(2587..3669)
                     /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
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISIVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3631..3650
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3808..3813,3814..3829,3830..3836)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3844..3860
                     /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)."
     RBS             4000..4008
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(4431..4450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4550..4572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4610..4628)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4696..4800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4801..4869,4870..5661)
                     /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(5019..5038)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5832..6420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6321..6340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
       61 ctttcctgcg ttatcagggg attccttaag gtatactttc cgctgcataa ccctgcttcg
      121 gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt
      181 gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag
      241 gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg
      301 cggtgctcaa cgggaatcct gctctgcgag gctggccgat aagctctgat accgctcgcc
      361 gcagccgaac gaccgagcgc agcgggtcag tgagcgagga agcggaagag cgcccaatac
      421 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagcgcccaa acataacagg
      481 aagaaaaatg ccccgctgtg ggcggacaaa atagttggga actgggaggg gtggaaatgg
      541 agtttttaag gattatttag ggaagagtga caaaatagat gggaactggg tgtagcgtcg
      601 taagctaata cgaaaattaa aaatgacaaa atagtttgga actagatttc acttatctgg
      661 ttggcctgca aggccttcgc gaggtaccgg gcccaagctt ctcgaggaat tcctgcagcc
      721 cgggggatcc actagtgagc tcatgcatga tcgaattagc ttcaaaagcg ctctgaagtt
      781 cctatacttt ctagagaata ggaacttcgg aataggaact tcaagatccc ctgattccct
      841 ttgtcaacag caatggatcg aattgacata agcctgttcg gttcgtaaac tgtaatgcaa
      901 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
      961 cgcagtggcg gttttcatgg cttgttatga ctgttttttt gtacagtcta tgcctcgggc
     1021 atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga
     1081 tgttacgcag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg
     1141 gctcaagtat gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc
     1201 gggctgctct tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc
     1261 agccggactc cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg
     1321 ccttcgacca agaagcggtt gttggcgctc tcgcggctta cgttctgccc aagtttgagc
     1381 agccgcgtag tgagatctat atctatgatc tcgcagtctc cggcgagcac cggaggcagg
     1441 gcattgccac cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg
     1501 tgatctacgt gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg
     1561 gcatacggga agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg
     1621 ttcaagccga gatcggcttc ccggccgcgg agttgttcgg taaattgtca caacgccgcg
     1681 gccaattcga tctagaattc cattgagtaa gtttttaagc acatcagctt caaaagcgct
     1741 ctgaagttcc tatactttct agagaatagg aacttcggaa taggtacttc aagatcccca
     1801 attcgatcgt ccgggccgca agctcctagc ggcggatttg tcctactcag gagagcgttc
     1861 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
     1921 tgatgcctca agctagagag tcattacccc aggcgtttaa gggcaccaat aactgcctta
     1981 aaaaaattac gccccgccct gccactcatc gcagtctagc ttggattctc accaataaaa
     2041 aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact
     2101 ggatctatca acaggagtcc aagctcagct aattaagcta gcttatcgat accgtcgacc
     2161 tcgaacccca cgcccctctt taatacgacg ggcaatttgc acttcagaaa atgaagagtt
     2221 tgctttagcc ataacaaaag tccagtatgc tttttcacag cataactgga ctgatttcag
     2281 tttacaacta ttctgtctag tttaagactt tattgtcata gtttagatct attttgttca
     2341 gtttaagact ttattgtccg cccacacctg cttacgcagg gcatccattt attactcaac
     2401 cgtaaccgat tttgccaggt tacgcggctg gtcctctagc tggcgtaata gcgaagaggc
     2461 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gatccgagcc
     2521 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2581 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2641 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     2701 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     2761 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     2821 gatataacat gagctgtctt cggtatcgtc gtatcccact atcgagatat ccgcaccaac
     2881 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     2941 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3001 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     3061 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     3121 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     3181 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     3241 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     3301 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     3361 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     3421 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     3481 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     3541 cgccatcgcc gcttccactt tttctcgcgt tttcgcagaa acgtggctgg cctggttcac
     3601 cacgcgagaa acggtctgat aagagacacc ggcatactct gcgacatcgt atagcgttac
     3661 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccacg
     3721 aaaggttttg caccattcga tgcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3781 tatttcacac cgcatatcga ggagctgttg acaattaatc atcggctcgt ataatgtgtg
     3841 gaattgtgag cggataacaa tttcacacgg ccggccaagg agaaaggcta tgaagcacaa
     3901 ccctctggtg gtgtgtctgc tcattatctg cattacgatt ctgacattca cactcctgac
     3961 ccgacaaacg ctctacgaac tgcggttccg ggacggtgat aaggaggttg ctgcgctcat
     4021 ggcctgcacg tccaggtaat cgcgagtcga caggaaggat aagaagaaca taaaaaatgg
     4081 cactattctc taaaatatta attttttatg tgattggtgt gaacatatcc tttgtcatta
     4141 tctggtttat ctcacatgag aaaacacata ttcgtttact tagtgcattc ctggtcggaa
     4201 taacctggcc aatgagtctg cctgtggcat tacttttttc tctcttttag gtcgacggcg
     4261 ccaggagacg cgtatgaacc tggtggatat cgccattctt atcctcaaac tcattgttgc
     4321 agcactgcaa ctgcttgatg ctgttctgaa atacctgaag taatacttaa ttaagggcaa
     4381 gcgcggggat tttccccgcg catttcccgg gtcagatggt gcactctcag tacaatctgc
     4441 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4501 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4561 atgtgtcaga ggttttcacc gtcgtcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4621 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4681 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4741 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4801 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4861 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4921 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4981 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5041 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5101 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5161 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5221 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5281 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5341 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5401 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5461 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5521 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5581 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5641 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5701 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5761 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5821 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5881 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5941 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6001 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6061 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6121 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6181 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6241 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6301 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6361 cacctctgac tcgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6421 aa
//
