LOCUS       sequence-427295-        4282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Medium copy thyA-infA plasmid containing bicistronic gfp-specR for 
            use in mouse work validation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4282)
  AUTHORS   Amrofell MB, Rengarajan S, Vo ST, Ramirez Tovar ES, LoBello L, 
            Dantas G, Moon TS
  TITLE     Engineering E. coli strains using antibiotic-resistance-gene-free 
            plasmids.
  JOURNAL   Cell Rep Methods. 2023 Dec 18;3(12):100669. doi: 
            10.1016/j.crmeth.2023.100669. Epub 2023 Dec 11.
  PUBMED    38086386
REFERENCE   2  (bases 1 to 4282)
  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: "Cell Rep 
            Methods."; date: "2023-12-18"; volume: "3(12):100669. doi"; pages: "
            10.1016/j.crmeth.2023.100669. Epub 2023 Dec 11"
COMMENT     Sequence Label: pMBA337
FEATURES             Location/Qualifiers
     source          1..4282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(1229..1323)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        1350..1384
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        1387..1436
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             1502..2215
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1532..1560)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2155..2175
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2512..3303
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPVQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     terminator      3356..3427
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3443..3470
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3498..3515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3632..4177)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3712..3731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgacagcta gctcagtcct aggtataatg ctagctcgac cgctatttta gcccatatca
       61 gaggtaacac ctaatgaaac agtatttaga actgatgcaa aaagtgctcg acgaaggcac
      121 acagaaaaac gaccgtaccg gaaccggaac gctttccatt tttggtcatc agatgcgttt
      181 taacctgcaa gatggattcc cgctggtgac aactaaacgt tgccacctgc gttccatcat
      241 ccatgaactg ctgtggtttc tgcagggcga cactaacatt gcttatctac acgaaaacaa
      301 tgtcaccatc tgggacgaat gggccgatga aaacggcgac ctcgggccag tgtatggtaa
      361 acagtggcgc gcctggccaa cgccagatgg tcgtcatatt gaccagatca ctacggtact
      421 gaaccagctg aaaaacgacc cggattcgcg ccgcattatt gtttcagcgt ggaacgtagg
      481 cgaactggat aaaatggcgc tggcaccgtg ccatgcattc ttccagttct atgtggcaga
      541 cggcaaactc tcttgccagc tttatcagcg ctcctgtgac gtcttcctcg gcctgccgtt
      601 caacattgcc agctacgcgt tattggtgca tatgatggcg cagcagtgcg atctggaagt
      661 gggtgatttt gtctggaccg gtggcgacac gcatctgtac agcaaccata tggatcaaac
      721 tcatctgcaa ttaagccgcg aaccgcgtcc gctgccgaag ttgattatca aacgtaaacc
      781 cgaatccatc ttcgactacc gtttcgaaga ctttgagatt gaaggctacg atccgcatcc
      841 gggcattaaa gcgccggtgg ctatctaagg ttcaaattac ggtagtgata ccccagagga
      901 ttagatggcc aaagaagaca atattgaaat gcaaggtacc gttcttgaaa cgttgcctaa
      961 taccatgttc cgcgtagagt tagaaaacgg tcacgtggtt actgcacaca tctccggtaa
     1021 aatgcgcaaa aactacatcc gcatcctgac gggcgacaaa gtgactgttg aactgacccc
     1081 gtacgacctg agcaaaggcc gcattgtctt ccgtagtcgc tgaagcctca aagtaatcaa
     1141 gccctggtaa ctgccagggc ttttttattt catctttgaa tcaacatatt aaaattcatc
     1201 gaatggcatc cttgctaaca gtgcttgtat tctcaccaat aaaaaacgcc cggcggcaac
     1261 cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga
     1321 gtccaagcga gctctcgaac cccagagtct tgacagctag ctcagtccta ggtataatgc
     1381 tagcagctgt caccggatgt gctttccggt ctgatgagtc cgtgaggacg aaacagcctc
     1441 tacaaataat tttgtttaac catccctata acaaaaaatc agcgcggcaa agtaggtaga
     1501 gatgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
     1561 tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg
     1621 aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact
     1681 tgtcactact ttgacttatg gtgttcaatg cttttcaaga tacccagatc atatgaaacg
     1741 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt
     1801 caaagatgac gggaactata agacacgtgc tgaagtcaag tttgaaggtg atacacttgt
     1861 taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa
     1921 gttggaatac aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg
     1981 aatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga
     2041 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
     2101 cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct
     2161 tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat acaaaaggcc
     2221 tgcagcaaac gacgaaaact acgcttaagt agcttaatcc tcattcggct tctccaataa
     2281 ggaggatcga taatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt
     2341 aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg
     2401 cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc
     2461 agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa
     2521 gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat
     2581 ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag
     2641 ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg
     2701 cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc
     2761 cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct
     2821 aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcggg tatcttcgag
     2881 ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt
     2941 gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt
     3001 gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag
     3061 cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg
     3121 ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggtccagta tcagcccgtc
     3181 atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca
     3241 gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa
     3301 taagcggaag agttaagacc attggatcca aactcgagta aggatctcca ggcatcaaat
     3361 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     3421 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataccta
     3481 gggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga
     3541 gcgaaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac
     3601 agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc
     3661 atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc
     3721 aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg
     3781 gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg
     3841 taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc
     3901 gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg
     3961 gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg
     4021 ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa
     4081 agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc
     4141 agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat
     4201 aaaatatttc tagatttcag tgcaatttat ctcttcaaat gtagcagagg ctagtgtaga
     4261 tcgctactag agcgcactga cc
//
