LOCUS       sequence-218394-        4978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP under the control of Pveg promoter and it is 
            integrated into AmyE locus. Used to test correct transformation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4978)
  AUTHORS   Wicke N, Radford D, Verrone V, Wipat A, French CE
  TITLE     BacilloFlex: A modular DNA assembly toolkit for Bacillus subtilis 
            synthetic biology
  JOURNAL   bioRxiv 185108
REFERENCE   2  (bases 1 to 4978)
  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: "bioRxiv 
            185108"
COMMENT     Sequence Label: pTU2-AmyE-Pveg-eGFP
FEATURES             Location/Qualifiers
     source          1..4978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..22
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             678..1391
                     /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="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(708..736)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1331..1351
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1407..1478
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1494..1521
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2952..3009
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(3033..3050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3204..3792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3284..3303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3975..4069)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4090..4749)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4664..4683
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     4911..4929
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4933..4976)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattcgcgg ccgcttctag aggtctcact atatcttttg caaaacgatt caaaacctct
       61 ttactgccgt tattcgctgg atttttattg ctgtttcatt tggttctggc aggaccggcg
      121 gctgcgagtg ctgaaacggc gaacaaatcg aatgagctta cagcaccgtc gatcaaaagc
      181 ggaaccattc ttcatgcatg gaattggtcg ttcaatacgt taaaacacaa tatgaaggat
      241 attcatgatg caggatatac agccattcag acatctccga ttaaccaagt aaaggaaggg
      301 aatcaaggag ataaaagcat gtcgaactgg tactggctgt atcagccgac atcgtatcaa
      361 attggcaacc gttacttagg tactgaacaa gaatttaaag aaatgtgtgc agccgctgaa
      421 gaatatggca taaaggtcat tgttgacgcg gtcatcaatc ataccaccag tgattatgcc
      481 gcgatttcca atgaggttaa gagtattcca aactggacac atggaaacac acaaattaaa
      541 aactggtctt gccctataat tttgtcaaaa taattttatt gacaacgtct tattaacgtt
      601 gatataattt aaattttatt tgacaaaaat gggctcgtgt tgtacaataa atgtgtacat
      661 attaagagga ggagcatatg cgtaaaggag aagaactttt cactggagtt gtcccaattc
      721 ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag
      781 gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg
      841 ttccatggcc aacacttgtc actactttcg gttatggtgt tcaatgcttt gcgagatacc
      901 cagatcatat gaaacagcat gactttttca agagtgccat gcccgaaggt tatgtacagg
      961 aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg
     1021 aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa
     1081 acattcttgg acacaaattg gaatacaact ataactcaca caatgtatac atcatggcag
     1141 acaaacaaaa gaatggaatc aaagttaact tcaaaattag acacaacatt gaagatggaa
     1201 gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt
     1261 taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga
     1321 gagatcacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg
     1381 aactatacaa ataatcgacc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     1441 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     1501 cttcgggtgg gcctttctgc gtttatatgt tccggcaccc attagttcaa caaacgaaaa
     1561 ttggataaag tgggatattt ttaaaatata tatttatgtt acaataatat tgacttttaa
     1621 aaaaggattg attctaatga agaaagcaga caagtaagcc tcctaaattc actttagata
     1681 aaaatttagg aggcatatca aatgaacttt aataaaattg atttagacaa ttggaagaga
     1741 aaagagatat ttaatcatta tttgaaccaa caaacgactt ttagtataac cacagaaatt
     1801 gatattagtg ttttataccg aaacataaaa caagaaggat ataaatttta ccctgcattt
     1861 attttcttag tgacaagggt gataaactca aatacagctt ttagaactgg ttacaatagc
     1921 gacggagagt taggttattg ggataagtta gagccacttt atacaatttt tgatggtgta
     1981 tctaaaacat tctctggtat ttggactcct gtaaagaatg acttcaaaga gttttatgat
     2041 ttataccttt ctgatgtaga gaaatataat ggttcgggga aattgtttcc caaaacacct
     2101 atacctgaaa atgctttttc tctttctatt attccatgga cttcatttac tgggtttaac
     2161 ttaaatatca ataataatag taattacctt ctacccatta ttacagcagg aaaattcatt
     2221 aataaaggta attcaatata tttaccgcta tctttacagg tacatcattc tgtttgtgat
     2281 ggttatcatg caggattgtt tatgaactct attcaggaat tgtcagatag gcctaatgac
     2341 tggcttttat aatatgagat aatgccgact gtacttttta cagtcggaag ttaggctggg
     2401 cggtgatagc ttctcgttca ggcagtacgc ctcttttctt ttccagacct gagggaggcg
     2461 gaaatggtgt gaggttcccg gggaaaagcc aaataggcga tcgcgggagt gctttatttg
     2521 aagatcaggc tatcactgcg gtcaatagat ttcacaatgt gatggctgga cagcctgagg
     2581 aactctcgaa cccgaatgga aacaaccaga tatttatgaa tcagcgcggc tcacatggcg
     2641 ttgtgctggc aaatgcaggt tcatcctctg tctctatcaa tacggcaaca aaattgcctg
     2701 atggcaggta tgacaataaa gctggagcgg gttcatttca agtgaacgat ggtaaactga
     2761 caggcacgat caatgccagg tctgtagctg tgctttatcc tgatgatatt gcaaaagcgc
     2821 ctcatgtttt ccttgagaat tacaaaacag gtgtaacaca ttctttcaat gatcaactga
     2881 cgattacctt gcgtgcagat gcgaatacaa caaaagccgt ttatcaaatc ttaggtacag
     2941 agaccctgca gtccggcaaa aaagggcaag gtgtcaccac cctgcccttt ttctttaaaa
     3001 ccgaaaagat tacttcgcgt tatgcaggct tcctcgctca ctgactcgct gcgctcggtc
     3061 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3181 aaaaaggccg cgttgctggc gtttttccac aggctccgcc cccctgacga gcatcacaaa
     3241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3961 agctcgaggc ttggattctc accaataaaa aacgcccggc ggcaaccgag cgttctgaac
     4021 aaatccagat ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc
     4081 gatatcaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
     4141 tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag
     4201 caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc
     4261 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgacacgaa
     4321 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
     4381 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
     4441 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
     4501 caccagctca ccgtctttca ttgccatacg aaattccgga tgagcattca tcaggcgggc
     4561 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
     4621 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     4681 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     4741 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
     4801 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgcccga tcaactcgag
     4861 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     4921 tcacgaggca gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctg
//
