LOCUS       sequence-419160-        8530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for biosynthetic pathway. pTac-phiC31 integrase. This 
            material contains 2 plasmids. For more information look under the 
            ""Depositor Comments"" section below..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8530)
  AUTHORS   Patel JR, Oh J, Wang S, Crawford JM, Isaacs FJ
  TITLE     Cross-kingdom expression of synthetic genetic elements promotes 
            discovery of metabolites in the human microbiome.
  JOURNAL   Cell. 2022 Apr 28;185(9):1487-1505.e14. doi: 
            10.1016/j.cell.2022.03.008. Epub 2022 Apr 1.
  PUBMED    35366417
REFERENCE   2  (bases 1 to 8530)
  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"; doi: 
            "10.1016/j.cell.2022.03.008"; journalName: "Cell"; date: 
            "2022-04-28- 28"; volume: "185"; issue: "9"; pages: "1487-1505.e14"
COMMENT     Sequence Label: pPath-pTac-integrase
FEATURES             Location/Qualifiers
     source          1..8530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..36
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        292..635
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             666..1475
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDVSD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(733..752)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1481..1678
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             1815..1832
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1885..1903)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1899..1946
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1965..1984
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1965..1983
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2013..2458
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2459..2545,2546..3880)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     rep_origin      3996..4384
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     promoter        join(4510..4515,4516..4531,4532..4538)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4542..4565
                     /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)."
     CDS             4652..6469
                     /codon_start=1
                     /product="integrase from phage phi-C31"
                     /label=phage phi-C31 integrase
                     /translation="MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDG
                     GRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIP
                     IVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQREL
                     GGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWRE
                     IKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRIL
                     RDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQA
                     WLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPG
                     QHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA
                     NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAE
                     APKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPI
                     EKRASITWAKPPTDDDEDDAQDGTEDVAA"
     promoter        complement(6470..6488)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6471..6488)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        6489..6580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6581..6649,6650..7441)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MGIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     oriT            7632..7741
                     /note="incP origin of transfer"
ORIGIN
        1 gggtgccagg gcgtgcccgt gggctccccg ggcgcgtata gggataacag ggtaatacgt
       61 caaattctat cataattgtg gtttcaaaat cggctccgtc gatactatgt tatacgccaa
      121 ctttgaaaac aactttgaaa aggctgtttt ctgtatttaa ggttttagaa tgcaaggaac
      181 agtgaattgg agttcgtctt gttataatta gcttcttggg gtatctttaa atactgtaga
      241 aaaagaaatt gcataccttt gttcctcggt tatatgtttg ctcatctgca agacatggag
      301 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt
      361 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat
      421 ggccgcacgg cgcgaagcaa aaattacggc acctcgctgc agacctgcga gcagggaaac
      481 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa
      541 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga
      601 tacagttctc acatcacatc cgaacataaa caaccaaaaa tttgtaatta agaaggagtg
      661 attacatggg taaggaaaag actcacgttt cgaggccgcg attaaattcc aacatggatg
      721 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
      781 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
      841 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
      901 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
      961 tccccggcaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     1021 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     1081 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     1141 ttgatgtgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     1201 aaatgcataa gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     1261 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     1321 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     1381 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     1441 tgcagtttca tttgatgctc gatgagtttt tctaatcagt actgacaata aaaagattct
     1501 tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc tattttaatc
     1561 aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgtccag atgcgaagtt
     1621 aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg ctatactgct
     1681 ctagcataac cccgcggggc ctctttcggg gatctcgcgg ggttttttgc tgaaagaagc
     1741 ttcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttatgtt gttgtcgctg
     1801 cggccgcact cgagcaccac caccaccacc actgggatcc ggctgctaac aaagcccgaa
     1861 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1921 ctaaacgggt cttgaggggt tttttgctga aggccatcat ggcctaatac gactcactat
     1981 agggagacca atgtgatgga cactgagacc tccacatata cctgccgttc actattattt
     2041 agtgaaatga gatattatga tattttctga attgtgatta aaaaggcaac tttatgccca
     2101 tgcaacagaa actataaaaa atacagagaa tgaaaagaaa cagatagatt ttttagttct
     2161 ttaggcccgt agtctgcaaa tccttttatg attttctatc aaacaaaaga ggaaaataga
     2221 ccagttgcaa tccaaacgag agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt
     2281 tgttactgat aaagcaggca agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg
     2341 tcacccctta catattttag gtcttttttt attgtgcgta actaacttgc catcttcaaa
     2401 caggagggct ggaagaagca gaccgctaac acagtacata aaaaaggaga catgaacgat
     2461 gaacatcaaa aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc
     2521 aggaggcgca actcaagcgt ttgcgaaaga aacgaaccaa aagccatata aggaaacata
     2581 cggcatttca catattacac gccatgatat gctgcaaatc cctgaacagc aaaaaaatga
     2641 aaaatatcaa gttcctgagt tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg
     2701 cctggacgtt tgggacagct ggccattaca aaacgctgac ggcactgtcg caaactatca
     2761 cggctaccac atcgtctttg cattagccgg agatcctaaa aatgcggatg acacatcgat
     2821 ttacatgttc tatcaaaaag tcggcgaaac ttctattgac agctggaaaa acgctggccg
     2881 cgtctttaaa gacagcgaca aattcgatgc aaatgattct atcctaaaag accaaacaca
     2941 agaatggtca ggttcagcca catttacatc tgacggaaaa atccgtttat tctacactga
     3001 tttctccggt aaacattacg gcaaacaaac actgacaact gcacaagtta acgtatcagc
     3061 atcagacagc tctttgaaca tcaacggtgt agaggattat aaatcaatct ttgacggtga
     3121 cggaaaaacg tatcaaaatg tacagcagtt catcgatgaa ggcaactaca gctcaggcga
     3181 caaccatacg ctgagagatc ctcactacgt agaagataaa ggccacaaat acttagtatt
     3241 tgaagcaaac actggaactg aagatggcta ccaaggcgaa gaatctttat ttaacaaagc
     3301 atactatggc aaaagcacat cattcttccg tcaagaaagt caaaaacttc tgcaaagcga
     3361 taaaaaacgc acggctgagt tagcaaacgg cgctctcggt atgattgagc taaacgatga
     3421 ttacacactg aaaaaagtga tgaaaccgct gattgcatct aacacagtaa cagatgaaat
     3481 tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac ctgttcactg actcccgcgg
     3541 atcaaaaatg acgattgacg gcattacgtc taacgatatt tacatgcttg gttatgtttc
     3601 taattcttta actggcccat acaagccgct gaacaaaact ggccttgtgt taaaaatgga
     3661 tcttgatcct aacgatgtaa cctttactta ctcacacttc gctgtacctc aagcgaaagg
     3721 aaacaatgtc gtgattacaa gctatatgac aaacagagga ttctacgcag acaaacaatc
     3781 aacgtttgcg ccaagtttcc tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga
     3841 cagcatcctt gaacaaggac aattaacagt taacaaataa ggtctctaga gccaccacag
     3901 tggggcctgt gaggccggac caaaacgaaa aaaggccccc ctttcgggag gcctcttttc
     3961 tggaatttgg taccgagtag gccggccggc gcgccgatct gaagatcagc agttcaacct
     4021 gttgatagta cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttaacgtact
     4081 aagctctcat gtttaacgaa ctaaaccctc atggctaacg tactaagctc tcatggctaa
     4141 cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttgaacaata aaattaatat
     4201 aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta taagaaaaaa aagaatatat
     4261 aaggctttta aagcctttaa ggtttaacgg ttgtggacaa caagccaggg atgtaacgca
     4321 ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga cacaggaaca cttaacggct
     4381 gacatggggc gcgcccagta gggataacag ggtaatgggt gccagggcgt gccccagggc
     4441 tccccgggcg cgtacctaat gagcgggctt ttttttaatt cccctacaaa attttagccg
     4501 ctagagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac
     4561 aattcaaatt tgcgcgccac attattattc atacctttgt ggaccgtatt acaaagagaa
     4621 gtgttaaatc aaacaaaaag gaggattaat catggacacg tacgcgggtg cttacgaccg
     4681 tcagtcgcgc gagcgcgaaa attcgagcgc agcaagccca gcgacacagc gtagcgccaa
     4741 cgaagacaag gcggccgacc ttcagcgcga agtcgagcgc gacgggggcc ggttcaggtt
     4801 cgtcgggcat ttcagcgaag cgccgggcac gtcggcgttc gggacggcgg agcgcccgga
     4861 gttcgaacgc atcctgaacg aatgccgcgc cgggcggctc aacatgatca ttgtctatga
     4921 cgtgtcgcgc ttctcgcgcc tgaaggtcat ggacgcgatt ccgattgtct cggaattgct
     4981 cgccctgggc gtgacgattg tttccactca ggaaggcgtc ttccggcagg gaaacgtcat
     5041 ggacctgatt cacctgatta tgcggctcga cgcgtcgcac aaagaatctt cgctgaagtc
     5101 ggcgaagatt ctcgacacga agaaccttca gcgcgaattg ggcgggtacg tcggcgggaa
     5161 ggcgccttac ggcttcgagc ttgtttcgga gacgaaggag atcacgcgca acggccgaat
     5221 ggtcaatgtc gtcatcaaca agcttgcgca ctcgaccact ccccttaccg gacccttcga
     5281 gttcgagccc gacgtaatcc ggtggtggtg gcgtgagatc aagacgcaca aacaccttcc
     5341 cttcaagccg ggcagtcaag ccgccattca cccgggcagc atcacggggc tttgtaagcg
     5401 catggacgct gacgccgtgc cgacccgggg cgagacgatt gggaagaaga ccgcttcaag
     5461 cgcctgggac ccggcaaccg ttatgcgaat ccttcgggac ccgcgtattg cgggcttcgc
     5521 cgctgaggtg atctacaaga agaagccgga cggcacgccg accacgaaga ttgagggtta
     5581 ccgcattcag cgcgacccga tcacgctccg gccggtcgag cttgattgcg gaccgatcat
     5641 cgagcccgct gagtggtatg agcttcaggc gtggttggac ggcagggggc gcggcaaggg
     5701 gctttcccgg gggcaagcca ttctgtccgc catggacaag ctgtactgcg agtgtggcgc
     5761 cgtcatgact tcgaagcgcg gggaagaatc gatcaaggac tcttaccgct gccgtcgccg
     5821 gaaggtggtc gacccgtccg cacctgggca gcacgaaggc acgtgcaacg tcagcatggc
     5881 ggcactcgac aagttcgttg cggaacgcat cttcaacaag atcaggcacg ccgaaggcga
     5941 cgaagagacg ttggcgcttc tgtgggaagc cgcccgacgc ttcggcaagc tcactgaggc
     6001 gcctgagaag agcggcgaac gggcgaacct tgttgcggag cgcgccgacg ccctgaacgc
     6061 ccttgaagag ctgtacgaag accgcgcggc aggcgcgtac gacggacccg ttggcaggaa
     6121 gcacttccgg aagcaacagg cagcgctgac gctccggcag caaggggcgg aagagcggct
     6181 tgccgaactt gaagccgccg aagccccgaa gcttcccctt gaccaatggt tccccgaaga
     6241 cgccgacgct gacccgaccg gccctaagtc gtggtggggg cgcgcgtcag tagacgacaa
     6301 gcgcgtgttc gtcgggctct tcgtagacaa gatcgttgtc acgaagtcga ctacgggcag
     6361 ggggcaggga acgcccatcg agaagcgcgc ttcgatcacg tgggcgaagc cgccgaccga
     6421 cgacgacgaa gacgacgccc aggacggcac ggaagacgta gcggcgtaat ctatagtgtc
     6481 acctaaattt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6541 accctgataa atgcttcaat aatattgaaa aaggaagagt atgggcattc agcattttcg
     6601 tgtggcgctg attccgtttt ttgcggcgtt ttgcctgccg gtgtttgcgc atccggaaac
     6661 cctggtgaaa gtgaaagatg cggaagatca actgggtgcg cgcgtgggct atattgaact
     6721 ggatctgaac agcggcaaaa ttctggaatc ttttcgtccg gaagaacgtt ttccgatgat
     6781 gagcaccttt aaagtgctgc tgtgcggtgc ggttctgagc cgtgtggatg cgggccagga
     6841 acaactgggc cgtcgtattc attatagcca gaacgatctg gtggaatata gcccggtgac
     6901 cgaaaaacat ctgaccgatg gcatgaccgt gcgtgaactg tgcagcgcgg cgattaccat
     6961 gagcgataac accgcggcga acctgctgct gacgaccatt ggcggtccga aagaactgac
     7021 cgcgtttctg cataacatgg gcgatcatgt gacccgtctg gatcgttggg aaccggaact
     7081 gaacgaagcg attccgaacg atgaacgtga taccaccatg ccggcagcaa tggcgaccac
     7141 cctgcgtaaa ctgctgacgg gtgagctgct gaccctggca agccgccagc aactgattga
     7201 ttggatggaa gcggataaag tggcgggtcc gctgctgcgt agcgcgctgc cggctggctg
     7261 gtttattgcg gataaaagcg gtgcgggcga acgtggcagc cgtggcatta ttgcggcgct
     7321 gggcccggat ggtaaaccga gccgtattgt ggtgatttat accaccggca gccaggcgac
     7381 gatggatgaa cgtaaccgtc agattgcgga aattggcgcg agcctgatta aacattggta
     7441 aaccgataca attaaaggct ccttttggag cctttttttt ttggacgacc cttgtccttt
     7501 tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt
     7561 ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa
     7621 cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc
     7681 actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg
     7741 gtcagtcaca gtggagccta gcactcgctc agcgtgacgg ctcagagcag aattcacgag
     7801 ccagaaatag taacttttgc ctaaatcaca aattgcaaaa tttaattgct tgcaaaaggt
     7861 cacatgctta taatcaactt ttttaaaaat ttaaaatact tttttatttt ttatttttaa
     7921 acataaatga aataatttat ttattgttta tgattaccga aacataaaac ctgctcaaga
     7981 aaaagaaact gttttgtcct tggaaaaaaa gcactaccta ggagcggcca aaatgccggc
     8041 ttacatttta tgttagctgg tggactgacg ccagaaaatg ttggtgatgc gcttagatta
     8101 aatggcgtta ttggtgttga tgtaagcgga ggtgtggaga caaatggtgt aaaagactct
     8161 aacaaaatag caaatttcgt caaaaatgct aagaaatagg ttattactga gtagtattta
     8221 tttaagtatt gtttgtgcac ttgcctgcaa gccttttgaa aagcaagcat aaaagatcta
     8281 aacataaaat ctgtaaaata acaagatgta aagataatgc taaatcattt ggctttttga
     8341 ttgattgtac aggaaaatat acatcgcagg gggttgactt ttaccatttc accgcaatgg
     8401 aattaaactt gttgaagaga atgttcacag gcgcatacgc tacaatgacc cgattcttgc
     8461 tagccgaatt ccagtcaggc tgctagcacc agagctacgt gaccgcagga ctagctccag
     8521 ctgagcgaca
//
