LOCUS       sequence-430401-        5602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy glmS-nadE plasmid containing kanamycin as antibiotic 
            resistance gene (ARG) for validation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5602)
  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 5602)
  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: pMBA324
FEATURES             Location/Qualifiers
     source          1..5602
                     /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"
     primer_bind     complement(2801..2818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(2973..3923)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMSGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3971..4193
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     primer_bind     complement(4436..4455)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4447..5262
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4447..5262
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4520..4539)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5466..5537
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5553..5580
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
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 gctgggcgac
      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 taaagcccga gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact gactcgctgc
     2821 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atcccgggca
     2881 gttttccctt tgatatctaa cggtgaacag ttgttctact tttgtttgtt agtcttgatg
     2941 cttcactgat agatacaaga gccataagaa cctcagatcc ttccgtattt agccagtatg
     3001 ttctctagtg tggttcgttg tttttgcgtg agccatgaga acgaaccatt gagatcatac
     3061 ttactttgca tgtcactcaa aaattttgcc tcaaaactgg tgagctgaat ttttgcagtt
     3121 aaagcatcgt gtagtgtttt tcttagtccg ttatgtaggt aggaatctga tgtaatggtt
     3181 gttggtattt tgtcaccact catttttatc tggttgttct caagttcggt tacgagatcc
     3241 atttgtctat ctagttcaac ttggaaaatc aacgtatcag tcgggcggcc tcgcttatca
     3301 accaccaatt tcatattgct gtaagtgttt aaatctttac ttattggttt caaaacccat
     3361 tggttaagcc ttttaaactc atggtagtta ttttcaagca ttaacatgaa cttaaattca
     3421 tcaaggctaa tctctatatt tgccttgtga gttttctttt gtgttagttc ttttaataac
     3481 cactcataaa tcctcataga gtatttgttt tcaaaagact taacatgttc cagattatat
     3541 tttatgaatt tttttaactg gaaaagataa ggcaatatct cttcactaaa aactaattct
     3601 aatttttcgc ttgagaactt ggcatagttt gtccactgga aaatctcaaa gcctttaacc
     3661 aaaggattcc tgatttccac agttctcgtc atcagctctc tggttgcttt agctaataca
     3721 ccataagcat tttccctact gatgttcatc atctgagcgt attggttata agtgaacgat
     3781 accgtccgtt ctttccttgt agggttttca atcgtggggt tgagtagtgc cacacagcat
     3841 aaaattagct tggtttcatg ctccgttaag tcatagcgac taatcgctag ttcatttgct
     3901 ttgaaaacaa ctaattcaga catacatctc aattggtcta ggtgatttta atcactatac
     3961 caattgagat gggctagtca atgataatta catgtccttt tcctttgagt tgtgggtatc
     4021 tgtaaattct gctagacctt tgctggaaaa cttgtaaatt ctgctagacc ctctgtaaat
     4081 tccgctagac ctttgtgtgt tttttttgtt tatattcaag tggttataat ttatagaata
     4141 aagaaagaat aaaaaaagat aaaaagaata gatcccagcc ctgtgtataa ctcactactt
     4201 tagtcagttc cgcagtatta caaaaggatg tcgcaaacgc tgtttgctcc tctacaaaac
     4261 agaccttaaa accctaaagg cttaagtagc accctcgcaa gctcgggcaa atcgctgaat
     4321 gttcgattta ttcaacaaag ccacgttgtg tctcaaaatc tctgatgtta cattgcacaa
     4381 gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     4441 ggtgttatga gccatattca acgggaaacg tcttgctcca ggccgcgatt aaattccaac
     4501 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     4561 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     4621 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     4681 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     4741 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     4801 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     4861 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     4921 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     4981 tggaaagaaa tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     5041 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     5101 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     5161 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     5221 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     5281 ttgtaacact ggcagagcat tacgctgact tgacgggact cgagattacc attattgaag
     5341 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5401 acaaataggg gttccgcgca catttcgaat tcgtgctagt gtagatcgct actagagcca
     5461 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     5521 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     5581 tttatacaat tgtctagata cc
//
