LOCUS       sequence-433358-        3822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High copy thyA-infA plasmid containing gfp and no antibiotic 
            resistance gene (ARG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3822)
  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 3822)
  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: pMBA334
FEATURES             Location/Qualifiers
     source          1..3822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(601..619)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(800..1513)
                     /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(840..860)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1455..1483
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     misc_RNA        complement(1579..1628)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(1631..1665)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     rep_origin      1836..2424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2325..2344
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2578..2595
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(join(3795..3822,1..7))
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
ORIGIN
        1 ctgtcagggt cagctcattt cagaatattt gccagaaccg ttatgatgtc ggcgcaaaaa
       61 acattatcca gaacgggagt gcgccttgag cgacacgaat tatgcagtga tttacgacct
      121 gcacagccat accacagctt ccgatggctg cctgacgcca gaagcattgg tgcaccgtgc
      181 agtcgataag cccggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
      241 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
      301 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggagaggacc gctttcgctg
      361 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
      421 agccttcgtc actggtcccg ccaccaaacg tttcggcggg gatggacatc tacgggatct
      481 aatctggtta agcccatact ttaaaaatta ataaggacca aaacgaaaaa agacgctttt
      541 cagcgtctct tttctggaat ttggtaccga gtctggccgt cgttttacac ctagggtgat
      601 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
      661 caggtggcac ttttcgggga aatgtgcgcg gaacccgata tctggcgaaa atgagacgtt
      721 gatcggcacg taagaggaat ggtcttaact cttccgctta agctacttaa gcgtagtttt
      781 cgtcgtttgc tgcaggcctt ttgtatagtt catccatgcc atgtgtaatc ccagcagctg
      841 ttacaaactc aagaaggacc atgtggtctc tcttttcgtt gggatctttc gaaagggcag
      901 attgtgtgga caggtaatgg ttgtctggta aaaggacagg gccatcgcca attggagtat
      961 tttgttgata atggtctgct agttgaacgc ttccatcttc aatgttgtgt ctaattttga
     1021 agttaacttt gattccattc ttttgtttgt ctgccatgat gtatacattg tgtgagttat
     1081 agttgtattc caacttgtgt ccaagaatgt ttccatcttc tttaaaatca atacctttta
     1141 actcgattct attaacaagt gtatcacctt caaacttgac ttcagcacgt gtcttatagt
     1201 tcccgtcatc tttgaaaaat atagttcttt cctgtacata accttcgggc atggcactct
     1261 tgaaaaagtc atgccgtttc atatgatctg ggtatcttga aaagcattga acaccataag
     1321 tcaaagtagt gacaagtgtt ggccatggaa caggtagttt tccagtagtg caaataaatt
     1381 taagggtaag ttttccgtat gttgcatcac cttcaccctc tccactgaca gaaaatttgt
     1441 gcccattaac atcaccatct aattcaacaa gaattgggac aactccagtg aaaagttctt
     1501 ctcctttact catctctacc tactttgccg cgctgatttt ttgttatagg gatggttaaa
     1561 caaaattatt tgtagaggct gtttcgtcct cacggactca tcagaccgga aagcacatcc
     1621 ggtgacagct gctagcatta tacctaggac tgagctagct gtcaactgtc agaccaagtt
     1681 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1741 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1801 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1861 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt ggcggatcaa
     1921 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1981 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2041 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2101 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2161 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2221 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2281 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2341 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2401 tcaggggggc ggagcctgtg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2461 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2521 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2581 gagtcagtga gcgaggaagc ctgcagtcta gactcgagta aggatccagt tcacagtggg
     2641 aataaaaaag cccccggaat gatcttccgg gggcaggagc tattactact taccattgtc
     2701 ttgctcggct tcagcgacta cggaagacaa tgcggccttt gctcaggtcg tacggggtca
     2761 gttcaacagt cactttgtcg cccgtcagga tgcggatgta gtttttgcgc attttaccgg
     2821 agatgtgtgc agtaaccacg tgaccgtttt ctaactctac gcggaacatg gtattaggca
     2881 acgtttcaag aacggtacct tgcatttcaa tattgtcttc tttggccatc taatcctctg
     2941 gggtatcact accgtaattt gaaccttaga tagccaccgg cgctttaatg cccggatgcg
     3001 gatcgtagcc ttcaatctca aagtcttcga aacggtagtc gaagatggat tcgggtttac
     3061 gtttgataat caacttcggc agcggacgcg gttcgcggct taattgcaga tgagtttgat
     3121 ccatatggtt gctgtacaga tgcgtgtcgc caccggtcca gacaaaatca cccacttcca
     3181 gatcgcactg ctgcgccatc atatgcacca ataacgcgta gctggcaatg ttgaacggca
     3241 ggccgaggaa gacgtcacag gagcgctgat aaagctggca agagagtttg ccgtctgcca
     3301 catagaactg gaagaatgca tggcacggtg ccagcgccat tttatccagt tcgcctacgt
     3361 tccacgctga aacaataatg cggcgcgaat ccgggtcgtt tttcagctgg ttcagtaccg
     3421 tagtgatctg gtcaatatga cgaccatctg gcgttggcca ggcgcgccac tgtttaccat
     3481 acactggccc gaggtcgccg ttttcatcgg cccattcgtc ccagatggtg acattgtttt
     3541 cgtgtagata agcaatgtta gtgtcgccct gcagaaacca cagcagttca tggatgatgg
     3601 aacgcaggtg gcaacgttta gttgtcacca gcgggaatcc atcttgcagg ttaaaacgca
     3661 tctgatgacc aaaaatggaa agcgttccgg ttccggtacg gtcgtttttc tgtgtgcctt
     3721 cgtcgagcac tttttgcatc agttctaaat actgtttcat taggtgttac ctctgatgct
     3781 aaaatagcgg tcgagctagc attataccta ggactgagct ag
//
