LOCUS       sequence-446608-        4240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini-Tn7 GFP expression vector - pBG35 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4240)
  AUTHORS   Roghair Stroud MN, Vang DX, Halverson LJ
  TITLE     Optimized CRISPR Interference System for Investigating Pseudomonas 
            alloputida Genes Involved in Rhizosphere Microbiome Assembly.
  JOURNAL   ACS Synth Biol. 2024 Aug 20. doi: 10.1021/acssynbio.4c00312.
  PUBMED    39163848
REFERENCE   2  (bases 1 to 4240)
  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: "ACS Synth 
            Biol. 2024 Aug 20. doi: 10.1021/acssynbio.4c00312."
COMMENT     Sequence Label: pMRS-BG35-GFP
FEATURES             Location/Qualifiers
     source          1..4240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             166..174
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     misc_feature    910..966
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      1009..1103
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1405..1424)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1416..2231
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            2398..2506
                     /note="incP origin of transfer"
     rep_origin      2528..2916
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     mobile_element  2925..3090
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(3104..3190)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3225..3272
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(3402..3935)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4124..4129,4130..4146,4147..4152))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    4193..4240
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 agagcgcttt tgaagctaat tcgatcatgc atgagcgacg tcttaattaa tttatttgac
       61 atgcgtgatg tttagaatta taatttgggg acctagggcc caagttcact taaaaaggag
      121 atcaacaatg aaagcaattt tcgtactgaa acatcttaat catgctaagg aggttttcta
      181 atgatcatgg gaattcataa aggtgaagaa ctgttcaccg gtgttgttcc gatcctggtt
      241 gaactggatg gtgatgttaa cggccacaaa ttctctgttc gtggtgaagg tgaaggtgat
      301 gcaaccaacg gtaaactgac cctgaaattc atctgcacta ccggtaaact gccggttcca
      361 tggccgactc tggtgactac cctgacctat ggtgttcagt gtttttctcg ttacccggat
      421 cacatgaagc agcatgattt cttcaaatct gcaatgccgg aaggttatgt acaggagcgc
      481 accatttctt tcaaagacga tggcacctac aaaacccgtg cagaggttaa atttgaaggt
      541 gatactctgg tgaaccgtat tgaactgaaa ggcattgatt tcaaagagga cggcaacatc
      601 ctgggccaca aactggaata taacttcaac tcccataacg tttacatcac cgcagacaaa
      661 cagaagaacg gtatcaaagc taacttcaaa attcgccata acgttgaaga cggtagcgta
      721 cagctggcgg accactacca gcagaacact ccgatcggtg atggtccggt tctgctgccg
      781 gataaccact acctgtccac ccagtctaaa ctgtccaaag acccgaacga aaagcgcgac
      841 cacatggtgc tgctggagtt cgttactgca gcaggtatca cgcacggcat ggatgaactc
      901 tacaaataag aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc
      961 aagcttgcgg ccgcgtcgtg actgggaaaa ccctggcgac tagtcttgga ctcctgttga
     1021 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     1081 gggcgttttt tattggtgag aatccagcca cgcccctctt taatacgacg ggcaatttgc
     1141 acttcagaaa atgaagagtt tgctttagcc ataacaaaag tccagtatgc tttttcacag
     1201 cataactgga ctgatttcag tttacaacta ttctgtctag tttaagactt tattgtcata
     1261 gtttagatct attttgttca gtttaagact ttattgtccg cccacaattt aaatttgtgt
     1321 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     1381 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcag cgtgaaacga
     1441 gctgtagccg tccgcgtctg aacagcaaca tggatgcgga tctgtatggc tataaatggg
     1501 cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg tctgtatggc aaaccggatg
     1561 cgccggaact gtttctgaaa catggcaaag gcagcgtggc gaacgatgtg accgatgaaa
     1621 tggtgcgtct gaactggctg accgaattta tgccgctgcc gaccattaaa cattttattc
     1681 gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc gggcaaaacc gcgtttcagg
     1741 tgctggaaga atatccggat agcggcgaaa acattgtgga tgcgctggcc gtgtttctgc
     1801 gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa cagcgatcgt gtgtttcgtc
     1861 tggcccaggc gcagagccgt atgaacaacg gcctggtgga tgcgagcgat tttgatgatg
     1921 aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat gcataaactg ctgccgttta
     1981 gcccggatag cgtggtgacc cacggcgatt ttagcctgga taacctgatt ttcgatgaag
     2041 gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat tgcggatcgt tatcaggatc
     2101 tggccattct gtggaactgc ctgggcgaat ttagcccgag cctgcaaaaa cgtctgtttc
     2161 agaaatatgg cattgataat ccggatatga acaaactgca atttcatctg atgctggatg
     2221 aatttttcta ataattaatt ggaccgcggt ccgcgcgttg tccttttccg ctgcataacc
     2281 ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac
     2341 aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg
     2401 caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc
     2461 gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccgtagg ccggccggcg
     2521 cgccccatgt cagccgttaa gtgttcctgt gtcactcaaa attgctttga gaggctctaa
     2581 gggcttctca gtgcgttaca tccctggctt gttgtccaca accgttaaac cctaaaggct
     2641 ttaaaagcct tatatattct tttttttctt ataaaactta aaaccttaga ggctatttaa
     2701 gttgctgatt tatattaatt ttattgttca aacatgagag cttagtacgt gaaacatgag
     2761 agcttagtac gttagccatg agagcttagt acgttagcca tgagggttta gttcgttaaa
     2821 catgagagct tagtacgtta aacatgagag cttagtacgt gaaacatgag agcttagtac
     2881 gtactatcaa caggttgaac tgctgatctt cagatcggcg cgcctgtggg cggacaaaat
     2941 agttgggaac tgggaggggt ggaaatggag tttttaagga ttatttaggg aagagtgaca
     3001 aaatagatgg gaactgggtg tagcgtcgta agctaatacg aaaattaaaa atgacaaaat
     3061 agtttggaac tagatttcac ttatctggtt tctagggcgg cggatttgtc ctactcagga
     3121 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc
     3181 gttttatttg atgccgacgt ccggacgatc gaattgggga tcttgaagta cctattccga
     3241 agttcctatt ctctagaaag tataggaact tcagagcgct tttgaagctg atgtgcttaa
     3301 aaacttactc aatggaattc tagatcgaat tggccgcggc gttgtgacaa tttaccgaac
     3361 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     3421 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     3481 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     3541 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3601 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3661 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3721 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3781 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3841 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3901 ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3961 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     4021 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     4081 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     4141 ggcttatgtc aatccgatcc attgctgttg acaaagggaa tcaggggatc ttgaagttcc
     4201 tattccgaag ttcctattct ctagaaagta taggaacttc
//
