LOCUS       sequence-433355-        3236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Medium 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 3236)
  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 3236)
  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: pMBA333
FEATURES             Location/Qualifiers
     source          1..3236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..72
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(1266..1360)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        1387..1421
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        1424..1473
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             1539..2252
                     /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(1569..1597)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2192..2212
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2347..2418
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2434..2461
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(2489..2506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2623..3168)
                     /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(2703..2722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agaggctagt gtagatcgct actagagcgc actgaccctg acagctagct cagtcctagg
       61 tataatgcta gctcgaccgc tattttagcc catatcagag gtaacaccta atgaaacagt
      121 atttagaact gatgcaaaaa gtgctcgacg aaggcacaca gaaaaacgac cgtaccggaa
      181 ccggaacgct ttccattttt ggtcatcaga tgcgttttaa cctgcaagat ggattcccgc
      241 tggtgacaac taaacgttgc cacctgcgtt ccatcatcca tgaactgctg tggtttctgc
      301 agggcgacac taacattgct tatctacacg aaaacaatgt caccatctgg gacgaatggg
      361 ccgatgaaaa cggcgacctc gggccagtgt atggtaaaca gtggcgcgcc tggccaacgc
      421 cagatggtcg tcatattgac cagatcacta cggtactgaa ccagctgaaa aacgacccgg
      481 attcgcgccg cattattgtt tcagcgtgga acgtaggcga actggataaa atggcgctgg
      541 caccgtgcca tgcattcttc cagttctatg tggcagacgg caaactctct tgccagcttt
      601 atcagcgctc ctgtgacgtc ttcctcggcc tgccgttcaa cattgccagc tacgcgttat
      661 tggtgcatat gatggcgcag cagtgcgatc tggaagtggg tgattttgtc tggaccggtg
      721 gcgacacgca tctgtacagc aaccatatgg atcaaactca tctgcaatta agccgcgaac
      781 cgcgtccgct gccgaagttg attatcaaac gtaaacccga atccatcttc gactaccgtt
      841 tcgaagactt tgagattgaa ggctacgatc cgcatccggg cattaaagcg ccggtggcta
      901 tctaaggttc aaattacggt agtgataccc cagaggatta gatggccaaa gaagacaata
      961 ttgaaatgca aggtaccgtt cttgaaacgt tgcctaatac catgttccgc gtagagttag
     1021 aaaacggtca cgtggttact gcacacatct ccggtaaaat gcgcaaaaac tacatccgca
     1081 tcctgacggg cgacaaagtg actgttgaac tgaccccgta cgacctgagc aaaggccgca
     1141 ttgtcttccg tagtcgctga agcctcaaag taatcaagcc ctggtaactg ccagggcttt
     1201 tttatttcat ctttgaatca acatattaaa attcatcgaa tggcatcctt gctaacagtg
     1261 cttgtattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
     1321 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct ctcgaacccc
     1381 agagtcttga cagctagctc agtcctaggt ataatgctag cagctgtcac cggatgtgct
     1441 ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaaccat
     1501 ccctataaca aaaaatcagc gcggcaaagt aggtagagat gagtaaagga gaagaacttt
     1561 tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt
     1621 ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt
     1681 gcactactgg aaaactacct gttccatggc caacacttgt cactactttg acttatggtg
     1741 ttcaatgctt ttcaagatac ccagatcata tgaaacggca tgactttttc aagagtgcca
     1801 tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg aactataaga
     1861 cacgtgctga agtcaagttt gaaggtgata cacttgttaa tagaatcgag ttaaaaggta
     1921 ttgattttaa agaagatgga aacattcttg gacacaagtt ggaatacaac tataactcac
     1981 acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta
     2041 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccaa
     2101 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt
     2161 cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg
     2221 ggattacaca tggcatggat gaactataca aaaggcctgc agcaaacgac gaaaactacg
     2281 cttaagtagc ttaagcggaa gagttaagac cattggatcc aaactcgagt aaggatctcc
     2341 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2401 ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc
     2461 gtttatacct agggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     2521 gactgcggcg agcgaaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga
     2581 agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc
     2641 ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact
     2701 ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt
     2761 tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac
     2821 tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt
     2881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa
     2941 aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc
     3001 gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta
     3061 cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt
     3121 ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt
     3181 attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagc
//
