LOCUS       sequence-427333-        5092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Medium copy glmS-nadE plasmid containing spectinomycin as antibiotic
            resistance gene (ARG) for validation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5092)
  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 5092)
  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: pMBA325
FEATURES             Location/Qualifiers
     source          1..5092
                     /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(2869..2963)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3343..4134
                     /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      4166..4237
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4253..4280
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4308..4325)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4442..4987)
                     /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(4522..4541)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgacagcta gctcagtcct aggtataatg ctagcgtcaa tttagtagta gaccacaagg
       61 aggttgctta atgtgtggaa ttgttggcgc gatcgcgcaa cgtgatgtag cagaaatcct
      121 tcttgaaggt ttacgtcgtc tggaataccg cggatatgac tctgccggtc tggccgttgt
      181 tgatgcagaa ggtcatatga cccgcctgcg tcgcctcggt aaagtccaga tgctggcaca
      241 ggcagcggaa gaacatcctc tgcatggcgg cactggtatt gctcacactc gctggacgac
      301 ccacggtgaa ccttcagaag tgaatgcgca tccgcatgtt tctgaacaca ttgtggtggt
      361 gcataacggc atcatcgaaa accatgaacc gctgcgtgaa gagctaaaag cgcgtggcta
      421 taccttcgtt tctgaaaccg acaccgaagt gattgcccat ctggtgaact gggagctgaa
      481 acaaggcggg actctgcgtg aggccgttct gcgtgctatc ccgcagctgc gtggtgcgta
      541 cggtacagtg atcatggact cccgtcaccc ggataccctg ctggcggcac gttctggtag
      601 tccgctggtg attggcctgg ggatgggcga aaactttatc gcttctgacc agctggcgct
      661 gttgccggtg acccgtcgct ttatcttcct tgaagagggc gatattgcgg aaatcactcg
      721 ccgttcggta aacatcttcg ataaaactgg cgcggaagta aaacgtcagg atatcgaatc
      781 caatctgcaa tatgacgcgg gcgataaagg catttaccgt cactacatgc agaaagagat
      841 ctacgaacag ccgaacgcga tcaaaaacac ccttaccgga cgcatcagcc acggtcaggt
      901 tgatttaagc gagctgggac cgaacgccga cgaactgctg tcgaaggttg agcatattca
      961 gatcctcgcc tgtggtactt cttataactc cggtatggtt tcccgctact ggtttgaatc
     1021 gctagcaggt attccgtgcg acgtcgaaat cgcctctgaa ttccgctatc gcaaatctgc
     1081 cgtgcgtcgt aacagcctga tgatcacctt gtcacagtct ggcgaaaccg cggataccct
     1141 ggctggcctg cgtctgtcga aagagctggg ttaccttggt tcactggcaa tctgtaacgt
     1201 tccgggttct tctctggtgc gcgaatccga tctggcgcta atgaccaacg cgggtacaga
     1261 aatcggcgtg gcatccacta aagcattcac cactcagtta actgtgctgt tgatgctggt
     1321 ggcgaagctg tctcgcctga aaggtctgga tgcctccatt gaacatgaca tcgtgcatgg
     1381 tctgcaggcg ctgccgagcc gtattgagca gatgctgtct caggacaaac gcattgaagc
     1441 gctggcagaa gatttctctg acaaacatca cgcgctgttc ctgggccgtg gcgatcagta
     1501 cccaatcgcg ctggaaggcg cattgaagtt gaaagagatc tcttacattc acgctgaagc
     1561 ctacgctgct ggcgaactga aacacggtcc gctggcgcta attgatgccg atatgccggt
     1621 tattgttgtt gcaccgaaca acgaattgct ggaaaaactg aaatccaaca ttgaagaagt
     1681 tcgcgcgcgt ggcggtcagt tgtatgtctt cgccgatcag gatgcgggtt ttgtaagtag
     1741 cgataacatg cacatcatcg agatgccgca tgtggaagag gtgattgcac cgatcttcta
     1801 caccgttccg ctgcagctgc tggcttacca tgtcgcgctg atcaaaggca ccgacgttga
     1861 ccagccgcgt aacctggcaa aatcggttac ggttgagtaa gaacgatacc taatagaaaa
     1921 taggaattga caccaatgac attgcaacaa caaataataa aggcgctggg cgcaaaaccg
     1981 cagattaatg ctgaagagga aattcgtcgt agtatcgatt ttctgaaaag ctacctgcaa
     2041 acttatccgt tcattaaatc actggtgctc gggatcagcg gcggtcagga ctccacgctt
     2101 gccggtaagc tgtgccagat ggcgattaat gagctgcgcc aggaaaccgg caacgaatca
     2161 ctgcaattta ttgccgtacg cctgccctat ggtgtccagg ccgacgaaca agattgccag
     2221 gatgccatcg cctttattca accggatcgc gtattaaccg ttaatatcaa gggcgcggta
     2281 ctcgccagcg agcaggcatt gcgggaagca ggcattgaac tgagcgattt tgtccgtggc
     2341 aatgaaaaag cgcgtgagcg gatgaaagca caatatagca ttgcgggtat gaccagcggt
     2401 gtcgtggtgg gcaccgatca tgctgcagaa gctattaccg gattcttcac taaatatggt
     2461 gacggcggta cggacattaa tccgctgtat cgtctcaaca aacgtcaggg taaacagtta
     2521 ctggcggcat taggttgccc ggaacacctt tataagaaag cgccaacggc cgatcttgaa
     2581 gatgatcgcc cttctctgcc ggatgaagtg gcactcggcg tgacctatga caatatcgac
     2641 gactatctgg aagggaaaaa cgtacctgaa caggtcgcca gaacaataga gaactggtat
     2701 ctgaaaaccg aacataaacg ccgtccgcca attaccgttt tcgatgattt ctggaaaaag
     2761 taaagcctca aagtaatcaa gccctggtaa ctgccagggc ttttttattt catctttgaa
     2821 tcaacatatt aaaattcatc gaatggcatc cttgctaaca gtgcttgtat tctcaccaat
     2881 aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat
     2941 tactggatct atcaacagga gtccaagcga gctctcgaac cccagagtcc cagccaggac
     3001 agaaatgcct cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg
     3061 atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag
     3121 cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
     3181 gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca
     3241 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
     3301 gttacgcagc agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc
     3361 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg
     3421 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt
     3481 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
     3541 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gtgagattct ccgcgctgta
     3601 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa
     3661 ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg
     3721 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta
     3781 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     3841 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     3901 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     3961 gtcgctgccg actgggcaat ggagcgcctg ccggtccagt atcagcccgt catacttgaa
     4021 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     4081 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataagatcca
     4141 aactcgagta aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4201 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     4261 ttcgggtggg cctttctgcg tttataccta gggatatatt ccgcttcctc gctcactgac
     4321 tcgctacgct cggtcgttcg actgcggcga gcgaaaatgg cttacgaacg gggcggagat
     4381 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
     4441 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     4501 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc
     4561 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
     4621 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
     4681 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4741 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga
     4801 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg
     4861 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa
     4921 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg
     4981 atctcaagaa gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat
     5041 ctcttcaaat gtagcagagg ctagtgtaga tcgctactag agcgcactga cc
//
