LOCUS       sequence-230537-        7274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  allelic exchange vector with GFP merodiploid tracker and 
            temperature-sensitive origin of replication.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7274)
  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 7274)
  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: pAX1
FEATURES             Location/Qualifiers
     source          1..7274
                     /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"
     primer_bind     complement(3776..3795)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3895..3917
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3955..3973)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4041..4145
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4146..4214,4215..5006)
                     /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(4364..4383)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6054..6276
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             6324..7274
                     /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 ctcgagtgga
     3541 ggccggcccc taggacgact gcagcccgct tacgcagggc atccatttat tactcaaccg
     3601 taaccgattt tgccaggtta cgcggctggt cctctagctg gcgtaatagc gaagaggccc
     3661 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     3721 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     3781 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3841 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3901 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3961 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4021 gcacttttcg gggaaatgta cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4081 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4141 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4201 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4261 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4321 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4381 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4441 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4501 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4561 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4621 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4681 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4741 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4801 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4861 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4921 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4981 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5041 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5101 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5161 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5221 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5281 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5341 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5401 tgttaccagc cggttgtcag ccgttaagtg ttcctgtgtc actcaaaatt gctttgagag
     5461 gctctaaggg cttctcagtg cgttacatcc ctggcttgtt gtccacaacc gttaaacctt
     5521 aaaagctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5581 ttgatgatac cgctgcctta ctgggtgcat tagccagtct gaatgacctg tcacgggata
     5641 atccgaagtg gtcagactgg aaaatcagag ggcaggaact gctgaacagc aaaaagtcag
     5701 atagcaccac atagcagacc cgccataaaa cgccctgaga agcccgtgac gggcttttct
     5761 tgtattatgg gtagtttcct tgcatgaatc cataaaaggc gcctgtagtg ccatttaccc
     5821 ccattcactg ccagagccgt gagcgcagcg aactgaatgt cacgaaaaag acagcgactc
     5881 aggtgcctga tggtcggaga caaaaggaat attcagcgat ttgcccgatt gcggccgcaa
     5941 ccgagcttgc gagggtgcta cttaagcctt tagggtttta aggtctgttt tgtagaggag
     6001 caaacagcgt ttgcgacatc cttttgtaat actgcggaac tgactaaagt agtgagttat
     6061 acacagggct gggatctatt ctttttatct ttttttattc tttctttatt ctataaatta
     6121 taaccacttg aatataaaca aaaaaaacac acaaaggtct agcggaattt acagagggtc
     6181 tagcagaatt tacaagtttt ccagcaaagg tctagcagaa tttacagata cccacaactc
     6241 aaaggaaaag gactagtaat tatcattgac tagcccatct caattggtat agtgattaaa
     6301 atcacctaga ccaattgaga tgtatgtctg aattagttgt tttcaaagca aatgaactag
     6361 cgattagtcg ctatgactta acggagcatg aaaccaagct aattttatgc tgtgtggcac
     6421 tactcaaccc cacgattgaa aaccctacaa ggaaagaacg gacggtatcg ttcacttata
     6481 accaatacgt tcagatgatg aacatcagta gggaaaatgc ttatggtgta ttagctaaag
     6541 caaccagaga gctgatgacg agaactgtgg aaatcaggaa tcctttggtt aaaggctttg
     6601 agattttcca gtggacaaac tatgccaagt tctcaagcga aaaattagaa ttagttttta
     6661 gtgaagagat attgccttat cttttccagt taaaaaaatt cataaaatat aatctggaac
     6721 atgttaagtc ttttgaaaac aaatactcta tgaggattta tgagtggtta ttaaaagaac
     6781 taacacaaaa gaaaactcac aaggcaaata tagagattag ccttgatgaa tttaagttca
     6841 tgttaatgct tgaaaataac taccatgagt ttaaaaggct taaccaatgg gttttgaaac
     6901 caataagtaa agatttaaac acttacagca atatgaaatt ggtggttgat aagcgaggcc
     6961 gcccgactga tacgttgatt ttccaagttg aactagatag acaaatggat ctcgtaaccg
     7021 aacttgagaa caaccagata aaaatgaatg gtgacaaaat accaacaacc attacatcag
     7081 attcctacct acataacgga ctaagaaaaa cactacacga tgctttaact gcaaaaattc
     7141 agctcaccag ttttgaggca aaatttttga gtgacatgca aagtaagtat gatctcaatg
     7201 gttcgttctc atggctcacg caaaaacaac gaaccacact agagaacata ctggctaaat
     7261 acggaaggat ctga
//
