LOCUS       sequence-433361-        5464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy glmS-nadE plasmid containing mCherry and no antibiotic 
            resistance gene (ARG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5464)
  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 5464)
  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: pMBA327
FEATURES             Location/Qualifiers
     source          1..5464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(522..539)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(694..1644)
                     /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      1692..1914
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     promoter        2046..2080
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             2101..2109
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2115..2825
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2260..2278)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2528..2547
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3049..3120
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3136..3163
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        3186..3220
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
ORIGIN
        1 actcgccagc gagcaggcat tgcgggaagc aggcattgaa ctgagcgatt ttgtccgtgg
       61 caatgaaaaa gcgcgtgagc ggatgaaagc acaatatagc attgcgggta tgaccagcgg
      121 tgtcgtggtg ggcaccgatc atgctgcaga agctattacc ggattcttca ctaaatatgg
      181 tgacggcggt acggacatta atccgctgta tcgtctcaac aaacgtcagg gtaaacagtt
      241 actggcggca ttaggttgcc cggaacacct ttataagaaa gcgccaacgg ccgatcttga
      301 agatgatcgc ccttctctgc cggatgaagt ggcactcggc gtgacctatg acaatatcga
      361 cgactatctg gaagggaaaa acgtacctga acaggtcgcc agaacaatag agaactggta
      421 tctgaaaacc gaacataaac gccgtccgcc aattaccgtt ttcgatgatt tctggaaaaa
      481 gtaaagcccg agaaaagatt acttcgcgtt atgcaggctt cctcgctcac tgactcgctg
      541 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatcccgggc
      601 agttttccct ttgatatcta acggtgaaca gttgttctac ttttgtttgt tagtcttgat
      661 gcttcactga tagatacaag agccataaga acctcagatc cttccgtatt tagccagtat
      721 gttctctagt gtggttcgtt gtttttgcgt gagccatgag aacgaaccat tgagatcata
      781 cttactttgc atgtcactca aaaattttgc ctcaaaactg gtgagctgaa tttttgcagt
      841 taaagcatcg tgtagtgttt ttcttagtcc gttatgtagg taggaatctg atgtaatggt
      901 tgttggtatt ttgtcaccac tcatttttat ctggttgttc tcaagttcgg ttacgagatc
      961 catttgtcta tctagttcaa cttggaaaat caacgtatca gtcgggcggc ctcgcttatc
     1021 aaccaccaat ttcatattgc tgtaagtgtt taaatcttta cttattggtt tcaaaaccca
     1081 ttggttaagc cttttaaact catggtagtt attttcaagc attaacatga acttaaattc
     1141 atcaaggcta atctctatat ttgccttgtg agttttcttt tgtgttagtt cttttaataa
     1201 ccactcataa atcctcatag agtatttgtt ttcaaaagac ttaacatgtt ccagattata
     1261 ttttatgaat ttttttaact ggaaaagata aggcaatatc tcttcactaa aaactaattc
     1321 taatttttcg cttgagaact tggcatagtt tgtccactgg aaaatctcaa agcctttaac
     1381 caaaggattc ctgatttcca cagttctcgt catcagctct ctggttgctt tagctaatac
     1441 accataagca ttttccctac tgatgttcat catctgagcg tattggttat aagtgaacga
     1501 taccgtccgt tctttccttg tagggttttc aatcgtgggg ttgagtagtg ccacacagca
     1561 taaaattagc ttggtttcat gctccgttaa gtcatagcga ctaatcgcta gttcatttgc
     1621 tttgaaaaca actaattcag acatacatct caattggtct aggtgatttt aatcactata
     1681 ccaattgaga tgggctagtc aatgataatt acatgtcctt ttcctttgag ttgtgggtat
     1741 ctgtaaattc tgctagacct ttgctggaaa acttgtaaat tctgctagac cctctgtaaa
     1801 ttccgctaga cctttgtgtg ttttttttgt ttatattcaa gtggttataa tttatagaat
     1861 aaagaaagaa taaaaaaaga taaaaagaat agatcccagc cctgtgtata actcactact
     1921 ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg ctgtttgctc ctctacaaaa
     1981 cagaccttaa aaccctaaag gcttaagtag caccctcgca agctcgggca aatcgctgaa
     2041 tgttcttgac agctagctca gtcctaggta taatgctagc ttcttaatta cgtaggacca
     2101 taaggaggtt tcctatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     2161 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     2221 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     2281 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     2341 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     2401 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     2461 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     2521 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     2581 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     2641 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     2701 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     2761 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     2821 agtaagcgga agagttaaga ccatttcaga attggttaat tggttgtaac actggcagag
     2881 cattacgctg acttgacggg actcgagatt accattattg aagcatttat cagggttatt
     2941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3001 gcacatttcg aattcgtgct agtgtagatc gctactagag ccaggcatca aataaaacga
     3061 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     3121 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac aattgtctag
     3181 ataccctgac agctagctca gtcctaggta taatgctagc gtcaatttag tagtagacca
     3241 caaggaggtt gcttaatgtg tggaattgtt ggcgcgatcg cgcaacgtga tgtagcagaa
     3301 atccttcttg aaggtttacg tcgtctggaa taccgcggat atgactctgc cggtctggcc
     3361 gttgttgatg cagaaggtca tatgacccgc ctgcgtcgcc tcggtaaagt ccagatgctg
     3421 gcacaggcag cggaagaaca tcctctgcat ggcggcactg gtattgctca cactcgctgg
     3481 gcgacccacg gtgaaccttc agaagtgaat gcgcatccgc atgtttctga acacattgtg
     3541 gtggtgcata acggcatcat cgaaaaccat gaaccgctgc gtgaagagct aaaagcgcgt
     3601 ggctatacct tcgtttctga aaccgacacc gaagtgattg cccatctggt gaactgggag
     3661 ctgaaacaag gcgggactct gcgtgaggcc gttctgcgtg ctatcccgca gctgcgtggt
     3721 gcgtacggta cagtgatcat ggactcccgt cacccggata ccctgctggc ggcacgttct
     3781 ggtagtccgc tggtgattgg cctggggatg ggcgaaaact ttatcgcttc tgaccagctg
     3841 gcgctgttgc cggtgacccg tcgctttatc ttccttgaag agggcgatat tgcggaaatc
     3901 actcgccgtt cggtaaacat cttcgataaa actggcgcgg aagtaaaacg tcaggatatc
     3961 gaatccaatc tgcaatatga cgcgggcgat aaaggcattt accgtcacta catgcagaaa
     4021 gagatctacg aacagccgaa cgcgatcaaa aacaccctta ccggacgcat cagccacggt
     4081 caggttgatt taagcgagct gggaccgaac gccgacgaac tgctgtcgaa ggttgagcat
     4141 attcagatcc tcgcctgtgg tacttcttat aactccggta tggtttcccg ctactggttt
     4201 gaatcgctag caggtattcc gtgcgacgtc gaaatcgcct ctgaattccg ctatcgcaaa
     4261 tctgccgtgc gtcgtaacag cctgatgatc accttgtcac agtctggcga aaccgcggat
     4321 accctggctg gcctgcgtct gtcgaaagag ctgggttacc ttggttcact ggcaatctgt
     4381 aacgttccgg gttcttctct ggtgcgcgaa tccgatctgg cgctaatgac caacgcgggt
     4441 acagaaatcg gcgtggcatc cactaaagca ttcaccactc agttaactgt gctgttgatg
     4501 ctggtggcga agctgtctcg cctgaaaggt ctggatgcct ccattgaaca tgacatcgtg
     4561 catggtctgc aggcgctgcc gagccgtatt gagcagatgc tgtctcagga caaacgcatt
     4621 gaagcgctgg cagaagattt ctctgacaaa catcacgcgc tgttcctggg ccgtggcgat
     4681 cagtacccaa tcgcgctgga aggcgcattg aagttgaaag agatctctta cattcacgct
     4741 gaagcctacg ctgctggcga actgaaacac ggtccgctgg cgctaattga tgccgatatg
     4801 ccggttattg ttgttgcacc gaacaacgaa ttgctggaaa aactgaaatc caacattgaa
     4861 gaagttcgcg cgcgtggcgg tcagttgtat gtcttcgccg atcaggatgc gggttttgta
     4921 agtagcgata acatgcacat catcgagatg ccgcatgtgg aagaggtgat tgcaccgatc
     4981 ttctacaccg ttccgctgca gctgctggct taccatgtcg cgctgatcaa aggcaccgac
     5041 gttgaccagc cgcgtaacct ggcaaaatcg gttacggttg agtaagaacg atacctaata
     5101 gaaaatagga attgacacca atgacattgc aacaacaaat aataaaggcg ctgggcgcaa
     5161 aaccgcagat taatgctgaa gaggaaattc gtcgtagtat cgattttctg aaaagctacc
     5221 tgcaaactta tccgttcatt aaatcactgg tgctcgggat cagcggcggt caggactcca
     5281 cgcttgccgg taagctgtgc cagatggcga ttaatgagct gcgccaggaa accggcaacg
     5341 aatcactgca atttattgcc gtacgcctgc cctatggtgt ccaggccgac gaacaagatt
     5401 gccaggatgc catcgccttt attcaaccgg atcgcgtatt aaccgttaat atcaagggcg
     5461 cggt
//
