LOCUS       sequence-230535-        8578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  allelic exchange vector with GFP merodiploid tracker, 
            temperature-sensitive origin of replication, and TetR-controlled 
            kill switch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8578)
  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 8578)
  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: pAX2
FEATURES             Location/Qualifiers
     source          1..8578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            370..478
                     /note="incP origin of transfer"
     promoter        join(742..747,748..764,765..770)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             959..1492
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        1606..1708
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1775..1794)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             2430..2441
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2448..3161
                     /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="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2478..2506)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3101..3121
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3204..3298
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3401..3487
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             3934..3942
                     /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)"
     RBS             4059..4081
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4110..4128
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4135..4153
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        join(4174..4179,4180..4195,4196..4202)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             4239..4862
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4312..4331)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5080..5099)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5199..5221
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5259..5277)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5345..5449
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5450..5518,5519..6310)
                     /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(5668..5687)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7358..7580
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             7628..8578
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
       61 aactgttcac cgttacatat caaagggaaa actgtccata cccatgtaaa acttaaaacc
      121 ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag
      181 tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg
      241 gtttagttcg ttaaacatga gagcttagta cgttaaactt gagagcttag tacgtgaaac
      301 atgagagctt agtacgtact atcaacaggt tgaactgccc atgttctttc ctgcgttatc
      361 agagcttatc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
      421 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
      481 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
      541 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
      601 tgcagcggaa agtatacctt aaggaatccc ctgataccgc tcgccgcagc cgaacgaccg
      661 agcgcagcgg gtcagtgagc gaggaagcgg aagagaattc caagatcccc tgattccctt
      721 tgtcaacagc aatggatcga attgacataa gcctgttcgg ttcgtaaact gtaatgcaag
      781 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
      841 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg tacagtctat gcctcgggca
      901 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
      961 gttacgcagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaggtgg
     1021 ctcaagtatg ggcatcattc gcacatgtag gctcggccct gaccaagtca aatccatgcg
     1081 ggctgctctt gatcttttcg gtcgtgagtt cggagacgta gccacctact cccaacatca
     1141 gccggactcc gattacctcg ggaacttgct ccgtagtaag acattcatcg cgcttgctgc
     1201 cttcgaccaa gaagcggttg ttggcgctct cgcggcttac gttctgccca agtttgagca
     1261 gccgcgtagt gagatctata tctatgatct cgcagtctcc ggcgagcacc ggaggcaggg
     1321 cattgccacc gcgctcatca atctcctcaa gcatgaggcc aacgcgcttg gtgcttatgt
     1381 gatctacgtg caagcagatt acggtgacga tcccgcagtg gctctctata caaagttggg
     1441 catacgggaa gaagtgatgc actttgatat cgacccaagt accgccacct aactctagac
     1501 aatatcgata cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt
     1561 tgataccggg aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag
     1621 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttgtc
     1681 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
     1741 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     1801 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
     1861 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     1921 ggaattacgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
     1981 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
     2041 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
     2101 ggcctatttc cctaaagggg ttattgagaa tatgtttttc gtctcagcca atccctgggt
     2161 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     2221 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2281 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     2341 ctgcgatgaa ctagtctttg gcagtttatt cttgacatgt agtgaggggg ctggtataat
     2401 cacatagtac tgttcgggta cctagaatta aagaggagaa attaagcatg cgtaaaggag
     2461 aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc
     2521 acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta
     2581 aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc actactttcg
     2641 gttatggtgt tcaatgcttt gcgagatacc cagatcatat gaaacagcat gactttttca
     2701 agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa gatgacggga
     2761 actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt
     2821 taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact
     2881 ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagttaact
     2941 tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa
     3001 atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat
     3061 ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa
     3121 cagctgctgg gattacacat ggcatggatg aactatacaa ataagcgagc tcgtttaaac
     3181 gctagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     3241 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     3301 aagctagact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     3361 ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca
     3421 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     3481 gacaaatccg ccgctaggag tcgcgagttt aaaccccggg gtcgaccaga ctcgagaaat
     3541 gcgcggggaa aatccccgcg cttgccctta attaagtatt acttcaggta tttcagaaca
     3601 gcatcaagca gttgcagtgc tgcaacaatg agtttgagga taagaatggc gatatccacc
     3661 aggttcatac gcgtctcctg gcgccgtcga cctaaaagag agaaaaaagt aatgccacag
     3721 gcagactcat tggccaggtt attccgacca ggaatgcact aagtaaacga atatgtgttt
     3781 tctcatgtga gataaaccag ataatgacaa aggatatgtt cacaccaatc acataaaaaa
     3841 ttaatatttt agagaatagt gccatttttt atgttcttct tatccttcct gtcgactcgc
     3901 gattacctgg acgtgcaggc catgagcgca gcaacctcct tatcaccgtc ccggaaccgc
     3961 agttcgtaga gcgtttgtcg ggtcaggagt gtgaatgtca gaatcgtaat gcagataatg
     4021 agcagacaca ccaccagagg gttgtgcttc atagcctttc tccttcttaa agttaaacaa
     4081 aattatttct agaagctttg tgctcagtat ctctatcact gatagggatg tcaatctcta
     4141 tcactgatag ggaggccggc ccctagggag ctgttgacaa ttaatcatcg gctcgtataa
     4201 tgtgtggaac tcaagaacaa taatctaggt attacatcat gtctagatta gataaaagta
     4261 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     4321 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     4381 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     4441 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     4501 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     4561 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     4621 agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag
     4681 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     4741 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     4801 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtctt
     4861 aactgcagcc cgcttacgca gggcatccat ttattactca accgtaaccg attttgccag
     4921 gttacgcggc tggtcctcta gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4981 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     5041 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     5101 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5161 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5221 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5281 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5341 tgtacgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5401 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5461 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5521 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5581 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5641 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5701 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5761 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5821 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5881 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5941 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6001 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6061 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6121 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6181 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6241 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6301 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6361 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6421 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6481 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6541 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6601 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6661 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagccggttg
     6721 tcagccgtta agtgttcctg tgtcactcaa aattgctttg agaggctcta agggcttctc
     6781 agtgcgttac atccctggct tgttgtccac aaccgttaaa ccttaaaagc tcatgaccaa
     6841 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgttgatg ataccgctgc
     6901 cttactgggt gcattagcca gtctgaatga cctgtcacgg gataatccga agtggtcaga
     6961 ctggaaaatc agagggcagg aactgctgaa cagcaaaaag tcagatagca ccacatagca
     7021 gacccgccat aaaacgccct gagaagcccg tgacgggctt ttcttgtatt atgggtagtt
     7081 tccttgcatg aatccataaa aggcgcctgt agtgccattt acccccattc actgccagag
     7141 ccgtgagcgc agcgaactga atgtcacgaa aaagacagcg actcaggtgc ctgatggtcg
     7201 gagacaaaag gaatattcag cgatttgccc gattgcggcc gcaaccgagc ttgcgagggt
     7261 gctacttaag cctttagggt tttaaggtct gttttgtaga ggagcaaaca gcgtttgcga
     7321 catccttttg taatactgcg gaactgacta aagtagtgag ttatacacag ggctgggatc
     7381 tattcttttt atcttttttt attctttctt tattctataa attataacca cttgaatata
     7441 aacaaaaaaa acacacaaag gtctagcgga atttacagag ggtctagcag aatttacaag
     7501 ttttccagca aaggtctagc agaatttaca gatacccaca actcaaagga aaaggactag
     7561 taattatcat tgactagccc atctcaattg gtatagtgat taaaatcacc tagaccaatt
     7621 gagatgtatg tctgaattag ttgttttcaa agcaaatgaa ctagcgatta gtcgctatga
     7681 cttaacggag catgaaacca agctaatttt atgctgtgtg gcactactca accccacgat
     7741 tgaaaaccct acaaggaaag aacggacggt atcgttcact tataaccaat acgttcagat
     7801 gatgaacatc agtagggaaa atgcttatgg tgtattagct aaagcaacca gagagctgat
     7861 gacgagaact gtggaaatca ggaatccttt ggttaaaggc tttgagattt tccagtggac
     7921 aaactatgcc aagttctcaa gcgaaaaatt agaattagtt tttagtgaag agatattgcc
     7981 ttatcttttc cagttaaaaa aattcataaa atataatctg gaacatgtta agtcttttga
     8041 aaacaaatac tctatgagga tttatgagtg gttattaaaa gaactaacac aaaagaaaac
     8101 tcacaaggca aatatagaga ttagccttga tgaatttaag ttcatgttaa tgcttgaaaa
     8161 taactaccat gagtttaaaa ggcttaacca atgggttttg aaaccaataa gtaaagattt
     8221 aaacacttac agcaatatga aattggtggt tgataagcga ggccgcccga ctgatacgtt
     8281 gattttccaa gttgaactag atagacaaat ggatctcgta accgaacttg agaacaacca
     8341 gataaaaatg aatggtgaca aaataccaac aaccattaca tcagattcct acctacataa
     8401 cggactaaga aaaacactac acgatgcttt aactgcaaaa attcagctca ccagttttga
     8461 ggcaaaattt ttgagtgaca tgcaaagtaa gtatgatctc aatggttcgt tctcatggct
     8521 cacgcaaaaa caacgaacca cactagagaa catactggct aaatacggaa ggatctga
//
