LOCUS       sequence-218568-        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"
     CDS             657..1370
                     /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(687..715)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1310..1330
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1386..1457
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1473..1500
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2931..2988
                     /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(3012..3029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3183..3771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3263..3282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3954..4048)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4069..4728)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4643..4662
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     4890..4908
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4912..4955)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    join(4958..4978,1)
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 ggtctcacta tatcttttgc aaaacgattc aaaacctctt tactgccgtt attcgctgga
       61 tttttattgc tgtttcattt ggttctggca ggaccggcgg ctgcgagtgc tgaaacggcg
      121 aacaaatcga atgagcttac agcaccgtcg atcaaaagcg gaaccattct tcatgcatgg
      181 aattggtcgt tcaatacgtt aaaacacaat atgaaggata ttcatgatgc aggatataca
      241 gccattcaga catctccgat taaccaagta aaggaaggga atcaaggaga taaaagcatg
      301 tcgaactggt actggctgta tcagccgaca tcgtatcaaa ttggcaaccg ttacttaggt
      361 actgaacaag aatttaaaga aatgtgtgca gccgctgaag aatatggcat aaaggtcatt
      421 gttgacgcgg tcatcaatca taccaccagt gattatgccg cgatttccaa tgaggttaag
      481 agtattccaa actggacaca tggaaacaca caaattaaaa actggtcttg ccctataatt
      541 ttgtcaaaat aattttattg acaacgtctt attaacgttg atataattta aattttattt
      601 gacaaaaatg ggctcgtgtt gtacaataaa tgtgtacata ttaagaggag gagcatatgc
      661 gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg
      721 ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac
      781 ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca acacttgtca
      841 ctactttcgg ttatggtgtt caatgctttg cgagataccc agatcatatg aaacagcatg
      901 actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag
      961 atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata
     1021 gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg
     1081 aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca
     1141 aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt
     1201 atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt
     1261 ccacacaatc tgccctttcg aaagatccca acgaaaagag agatcacatg gtccttcttg
     1321 agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa taatcgacca
     1381 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     1441 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     1501 tttatatgtt ccggcaccca ttagttcaac aaacgaaaat tggataaagt gggatatttt
     1561 taaaatatat atttatgtta caataatatt gacttttaaa aaaggattga ttctaatgaa
     1621 gaaagcagac aagtaagcct cctaaattca ctttagataa aaatttagga ggcatatcaa
     1681 atgaacttta ataaaattga tttagacaat tggaagagaa aagagatatt taatcattat
     1741 ttgaaccaac aaacgacttt tagtataacc acagaaattg atattagtgt tttataccga
     1801 aacataaaac aagaaggata taaattttac cctgcattta ttttcttagt gacaagggtg
     1861 ataaactcaa atacagcttt tagaactggt tacaatagcg acggagagtt aggttattgg
     1921 gataagttag agccacttta tacaattttt gatggtgtat ctaaaacatt ctctggtatt
     1981 tggactcctg taaagaatga cttcaaagag ttttatgatt tatacctttc tgatgtagag
     2041 aaatataatg gttcggggaa attgtttccc aaaacaccta tacctgaaaa tgctttttct
     2101 ctttctatta ttccatggac ttcatttact gggtttaact taaatatcaa taataatagt
     2161 aattaccttc tacccattat tacagcagga aaattcatta ataaaggtaa ttcaatatat
     2221 ttaccgctat ctttacaggt acatcattct gtttgtgatg gttatcatgc aggattgttt
     2281 atgaactcta ttcaggaatt gtcagatagg cctaatgact ggcttttata atatgagata
     2341 atgccgactg tactttttac agtcggaagt taggctgggc ggtgatagct tctcgttcag
     2401 gcagtacgcc tcttttcttt tccagacctg agggaggcgg aaatggtgtg aggttcccgg
     2461 ggaaaagcca aataggcgat cgcgggagtg ctttatttga agatcaggct atcactgcgg
     2521 tcaatagatt tcacaatgtg atggctggac agcctgagga actctcgaac ccgaatggaa
     2581 acaaccagat atttatgaat cagcgcggct cacatggcgt tgtgctggca aatgcaggtt
     2641 catcctctgt ctctatcaat acggcaacaa aattgcctga tggcaggtat gacaataaag
     2701 ctggagcggg ttcatttcaa gtgaacgatg gtaaactgac aggcacgatc aatgccaggt
     2761 ctgtagctgt gctttatcct gatgatattg caaaagcgcc tcatgttttc cttgagaatt
     2821 acaaaacagg tgtaacacat tctttcaatg atcaactgac gattaccttg cgtgcagatg
     2881 cgaatacaac aaaagccgtt tatcaaatct taggtacaga gaccctgcag tccggcaaaa
     2941 aagggcaagg tgtcaccacc ctgccctttt tctttaaaac cgaaaagatt acttcgcgtt
     3001 atgcaggctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3061 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3181 tttttccaca ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gctcgaggct tggattctca
     3961 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag
     4021 gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt acgccccgcc
     4081 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
     4141 acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
     4201 atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
     4261 aaaactggtg aaactcaccc agggattggc tgacacgaaa aacatattct caataaaccc
     4321 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
     4381 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
     4441 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
     4501 tgccatacga aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
     4561 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
     4621 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
     4681 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt
     4741 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
     4801 gtgaaagttg gaacctctta cgtgcccgat caactcgagt gccacctgac gtctaagaaa
     4861 ccattattat catgacatta acctataaaa ataggcgtat cacgaggcag aatttcagat
     4921 aaaaaaaatc cttagctttc gctaaggatg atttctggaa ttcgcggccg cttctaga
//
