LOCUS       sequence-433364-        3952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low 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 3952)
  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 3952)
  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: pMBA332
FEATURES             Location/Qualifiers
     source          1..3952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     527..544
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(1670..1704)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(1727..1754)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(1770..1841)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2107..2820)
                     /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(2147..2167)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2762..2790
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     misc_RNA        complement(2886..2935)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(2938..2972)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     rep_origin      3104..3326
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             join(3374..3952,1..372)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRKMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 ataagtaaag atttaaacac ttacagcaat atgaaattgg tggttgataa gcgaggccgc
       61 ccgactgata cgttgatttt ccaagttgaa ctagatagaa aaatggatct cgtaaccgaa
      121 cttgagaaca accagataaa aatgaatggt gacaaaatac caacaaccat tacatcagat
      181 tcctacctac ataacggact aagaaaaaca ctacacgatg ctttaactgc aaaaattcag
      241 ctcaccagtt ttgaggcaaa atttttgagt gacatgcaaa gtaagtatga tctcaatggt
      301 tcgttctcat ggctcacgca aaaacaacga accacactag agaacatact ggctaaatac
      361 ggaaggatct gaggttctta tggctcttgt atctatcagt gaagcatcaa gactaacaaa
      421 caaaagtaga acaactgttc accgttagat atcaaaggga aaactgcccg ggattaccgc
      481 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
      541 cgaggaagcc tgcataacgc gaagtaatct tttctcgggc ttcagcgact acggaagaca
      601 atgcggcctt tgctcaggtc gtacggggtc agttcaacag tcactttgtc gcccgtcagg
      661 atgcggatgt agtttttgcg cattttaccg gagatgtgtg cagtaaccac gtgaccgttt
      721 tctaactcta cgcggaacat ggtattaggc aacgtttcaa gaacggtacc ttgcatttca
      781 atattgtctt ctttggccat ctaatcctct ggggtatcac taccgtaatt tgaaccttag
      841 atagccaccg gcgctttaat gcccggatgc ggatcgtagc cttcaatctc aaagtcttcg
      901 aaacggtagt cgaagatgga ttcgggttta cgtttgataa tcaacttcgg cagcggacgc
      961 ggttcgcggc ttaattgcag atgagtttga tccatatggt tgctgtacag atgcgtgtcg
     1021 ccaccggtcc agacaaaatc acccacttcc agatcgcact gctgcgccat catatgcacc
     1081 aataacgcgt agctggcaat gttgaacggc aggccgagga agacgtcaca ggagcgctga
     1141 taaagctggc aagagagttt gccgtctgcc acatagaact ggaagaatgc atggcacggt
     1201 gccagcgcca ttttatccag ttcgcctacg ttccacgctg aaacaataat gcggcgcgaa
     1261 tccgggtcgt ttttcagctg gttcagtacc gtagtgatct ggtcaatatg acgaccatct
     1321 ggcgttggcc aggcgcgcca ctgtttacca tacactggcc cgaggtcgcc gttttcatcg
     1381 gcccattcgt cccagatggt gacattgttt tcgtgtagat aagcaatgtt agtgtcgccc
     1441 tgcagaaacc acagcagttc atggatgatg gaacgcaggt ggcaacgttt agttgtcacc
     1501 agcgggaatc catcttgcag gttaaaacgc atctgatgac caaaaatgga aagcgttccg
     1561 gttccggtac ggtcgttttt ctgtgtgcct tcgtcgagca ctttttgcat cagttctaaa
     1621 tactgtttca ttaggtgtta cctctgatat gggctaaaat agcggtcgag ctagcattat
     1681 acctaggact gagctagctg tcagggtatc tagacaattg tataaacgca gaaaggccca
     1741 cccgaaggtg agccagtgtg actctagtag agagcgttca ccgacaaaca acagataaaa
     1801 cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc tctagtagcg
     1861 atctacacta gcacgaattc gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1921 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatggta
     1981 atctcgagtc ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt
     2041 ctgaaatggt cttaactctt ccgcttaagc tacttaagcg tagttttcgt cgtttgctgc
     2101 aggccttttg tatagttcat ccatgccatg tgtaatccca gcagctgtta caaactcaag
     2161 aaggaccatg tggtctctct tttcgttggg atctttcgaa agggcagatt gtgtggacag
     2221 gtaatggttg tctggtaaaa ggacagggcc atcgccaatt ggagtatttt gttgataatg
     2281 gtctgctagt tgaacgcttc catcttcaat gttgtgtcta attttgaagt taactttgat
     2341 tccattcttt tgtttgtctg ccatgatgta tacattgtgt gagttatagt tgtattccaa
     2401 cttgtgtcca agaatgtttc catcttcttt aaaatcaata ccttttaact cgattctatt
     2461 aacaagtgta tcaccttcaa acttgacttc agcacgtgtc ttatagttcc cgtcatcttt
     2521 gaaaaatata gttctttcct gtacataacc ttcgggcatg gcactcttga aaaagtcatg
     2581 ccgtttcata tgatctgggt atcttgaaaa gcattgaaca ccataagtca aagtagtgac
     2641 aagtgttggc catggaacag gtagttttcc agtagtgcaa ataaatttaa gggtaagttt
     2701 tccgtatgtt gcatcacctt caccctctcc actgacagaa aatttgtgcc cattaacatc
     2761 accatctaat tcaacaagaa ttgggacaac tccagtgaaa agttcttctc ctttactcat
     2821 ctctacctac tttgccgcgc tgattttttg ttatagggat ggttaaacaa aattatttgt
     2881 agaggctgtt tcgtcctcac ggactcatca gaccggaaag cacatccggt gacagctgct
     2941 agcattatac ctaggactga gctagctgtc aagaacattc agcgatttgc ccgagcttgc
     3001 gagggtgcta cttaagcctt tagggtttta aggtctgttt tgtagaggag caaacagcgt
     3061 ttgcgacatc cttttgtaat actgcggaac tgactaaagt agtgagttat acacagggct
     3121 gggatctatt ctttttatct ttttttattc tttctttatt ctataaatta taaccacttg
     3181 aatataaaca aaaaaaacac acaaaggtct agcggaattt acagagggtc tagcagaatt
     3241 tacaagtttt ccagcaaagg tctagcagaa tttacagata cccacaactc aaaggaaaag
     3301 gacatgtaat tatcattgac tagcccatct caattggtat agtgattaaa atcacctaga
     3361 ccaattgaga tgtatgtctg aattagttgt tttcaaagca aatgaactag cgattagtcg
     3421 ctatgactta acggagcatg aaaccaagct aattttatgc tgtgtggcac tactcaaccc
     3481 cacgattgaa aaccctacaa ggaaagaacg gacggtatcg ttcacttata accaatacgc
     3541 tcagatgatg aacatcagta gggaaaatgc ttatggtgta ttagctaaag caaccagaga
     3601 gctgatgacg agaactgtgg aaatcaggaa tcctttggtt aaaggctttg agattttcca
     3661 gtggacaaac tatgccaagt tctcaagcga aaaattagaa ttagttttta gtgaagagat
     3721 attgccttat cttttccagt taaaaaaatt cataaaatat aatctggaac atgttaagtc
     3781 ttttgaaaac aaatactcta tgaggattta tgagtggtta ttaaaagaac taacacaaaa
     3841 gaaaactcac aaggcaaata tagagattag ccttgatgaa tttaagttca tgttaatgct
     3901 tgaaaataac taccatgagt ttaaaaggct taaccaatgg gttttgaaac ca
//
