LOCUS       sequence-221660-        7272 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 7272)
  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 7272)
  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..7272
                     /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             2428..2439
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2446..3159
                     /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(2476..2504)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3099..3119
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3202..3296
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3399..3485
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3774..3793)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3893..3915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3953..3971)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4039..4143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4144..4212,4213..5004)
                     /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(4362..4381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6052..6274
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             6322..7272
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     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 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
     2161 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     2221 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2281 tcatcatgcc gtttgtgatg gcttccatgt cggcagatgc ttaatgaata caacagtact
     2341 gcgatgaact agtctttggc agtttattct tgacatgtag tgagggggct ggtataatca
     2401 catagtactg ttcgggtacc tagaattaaa gaggagaaat taagcatgcg taaaggagaa
     2461 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
     2521 aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa
     2581 tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcggt
     2641 tatggtgttc aatgctttgc gagataccca gatcatatga aacagcatga ctttttcaag
     2701 agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac
     2761 tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
     2821 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
     2881 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc
     2941 aaaattagac acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat
     3001 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct
     3061 gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca
     3121 gctgctggga ttacacatgg catggatgaa ctatacaaat aagcgagctc gtttaaacgc
     3181 tagcttaatt agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
     3241 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa
     3301 gctagactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc
     3361 ttaaacgcct ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt
     3421 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     3481 caaatccgcc gctaggagtc gcgagtttaa accccggggt cgaccagact cgagtggagg
     3541 ccggccccta ggacgactgc agcccgctta cgcagggcat ccatttatta ctcaaccgta
     3601 accgattttg ccaggttacg cggctggtcc tctagctggc gtaatagcga agaggcccgc
     3661 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     3721 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     3781 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3841 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3901 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3961 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4021 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4081 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4141 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4201 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4261 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4321 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4381 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4441 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4501 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4561 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4621 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4681 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4741 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4801 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4861 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4921 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4981 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5041 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5101 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5161 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5221 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5281 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     5341 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5401 ttaccagccg gttgtcagcc gttaagtgtt cctgtgtcac tcaaaattgc tttgagaggc
     5461 tctaagggct tctcagtgcg ttacatccct ggcttgttgt ccacaaccgt taaaccttaa
     5521 aagctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgtt
     5581 gatgataccg ctgccttact gggtgcatta gccagtctga atgacctgtc acgggataat
     5641 ccgaagtggt cagactggaa aatcagaggg caggaactgc tgaacagcaa aaagtcagat
     5701 agcaccacat agcagacccg ccataaaacg ccctgagaag cccgtgacgg gcttttcttg
     5761 tattatgggt agtttccttg catgaatcca taaaaggcgc ctgtagtgcc atttaccccc
     5821 attcactgcc agagccgtga gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag
     5881 gtgcctgatg gtcggagaca aaaggaatat tcagcgattt gcccgattgc ggccgcaacc
     5941 gagcttgcga gggtgctact taagccttta gggttttaag gtctgttttg tagaggagca
     6001 aacagcgttt gcgacatcct tttgtaatac tgcggaactg actaaagtag tgagttatac
     6061 acagggctgg gatctattct ttttatcttt ttttattctt tctttattct ataaattata
     6121 accacttgaa tataaacaaa aaaaacacac aaaggtctag cggaatttac agagggtcta
     6181 gcagaattta caagttttcc agcaaaggtc tagcagaatt tacagatacc cacaactcaa
     6241 aggaaaagga ctagtaatta tcattgacta gcccatctca attggtatag tgattaaaat
     6301 cacctagacc aattgagatg tatgtctgaa ttagttgttt tcaaagcaaa tgaactagcg
     6361 attagtcgct atgacttaac ggagcatgaa accaagctaa ttttatgctg tgtggcacta
     6421 ctcaacccca cgattgaaaa ccctacaagg aaagaacgga cggtatcgtt cacttataac
     6481 caatacgctc agatgatgaa catcagtagg gaaaatgctt atggtgtatt agctaaagca
     6541 accagagagc tgatgacgag aactgtggaa atcaggaatc ctttggttaa aggctttgag
     6601 attttccagt ggacaaacta tgccaagttc tcaagcgaaa aattagaatt agtttttagt
     6661 gaagagatat tgccttatct tttccagtta aaaaaattca taaaatataa tctggaacat
     6721 gttaagtctt ttgaaaacaa atactctatg aggatttatg agtggttatt aaaagaacta
     6781 acacaaaaga aaactcacaa ggcaaatata gagattagcc ttgatgaatt taagttcatg
     6841 ttaatgcttg aaaataacta ccatgagttt aaaaggctta accaatgggt tttgaaacca
     6901 ataagtaaag atttaaacac ttacagcaat atgaaattgg tggttgataa gcgaggccgc
     6961 ccgactgata cgttgatttt ccaagttgaa ctagatagac aaatggatct cgtaaccgaa
     7021 cttgagaaca accagataaa aatgaatggt gacaaaatac caacaaccat tacatcagat
     7081 tcctacctac gtaacggact aagaaaaaca ctacacgatg ctttaactgc aaaaattcag
     7141 ctcaccagtt ttgaggcaaa atttttgagt gacatgcaaa gtaagtatga tctcaatggt
     7201 tcgttctcat ggctcacgca aaaacaacga accacactag agaacatact ggctaaatac
     7261 ggaaggatct ga
//
