LOCUS       sequence-234472-        7868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad host range plasmid. Bacteria plasmid vector to test various 
            parts and devices. Expression of GFP and a Kanamycin/Neomycin 
            resistance gene in various cyanobacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7868)
  AUTHORS   Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepez J, Brahamsha B, 
            Palenik B, Peterson TC, Haerizadeh F, Golden SS, Golden JW
  TITLE     Broad-host-range vector system for synthetic biology and 
            biotechnology in cyanobacteria.
  JOURNAL   Nucleic Acids Res. 2014 Jul 29. pii: gku673.
  PUBMED    25074377
REFERENCE   2  (bases 1 to 7868)
  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: "Nucleic 
            Acids Res. 2014 Jul 29. pii: gku673."
COMMENT     Sequence Label: pAM4891 (pCV0003)
FEATURES             Location/Qualifiers
     source          1..7868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(397..1248)
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(1235..2074)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             complement(2585..3556)
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELSERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     oriT            4822..4909
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      5251..5645
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     terminator      complement(5808..5851)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(5993..6808)
                     /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"
     CDS             complement(5993..6808)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6716..6735
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6800..6819
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        join(6954..6959,6960..6975,6976..6982)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             7006..7026
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7036..7749
                     /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
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(7066..7094)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     7689..7709
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(7772..7796)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      7806..7833
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tctagaacaa tggaagtcct cgtctcgccc tcgaattagc ccgcttaatg agcgggcttt
       61 ttttgaatta attctcgcga gctggcacga caggtttccc gactggaaag cgggcagtga
      121 gcgcaacgca attaatgtaa gttagcgcga attgcaagct ggccgacgcg ctgggctacg
      181 tcttgctggc gttcgggagc agaagagcat acatctggaa gcaaagccag gaaagcggcc
      241 tatggagctg tgcggcagcg ctcagtaggc aatttttcaa aatattgtta agccttttct
      301 gagcatggta tttttcatgg tattaccaat tagcaggaaa ataagccatt gaatataaaa
      361 gataaaaatg tcttgtttac aatagagtgg ggggggtcag cctgccgcct tgggccgggt
      421 gatgtcgtac ttgcccgccg cgaactcggt taccgtccag cccagcgcga ccagctccgg
      481 caacgcctcg cgcacccgct ggcggcgctt gcgcatggtc gaaccactgg cctctgacgg
      541 ccagacatag ccgcacaagg tatctatgga agccttgccg gttttgccgg ggtcgatcca
      601 gccacacagc cgctggtgca gcaggcgggc ggtttcgctg tccagcgccc gcacctcgtc
      661 catgctgatg cgcacatgct ggccgccacc catgacggcc tgcgcgatca aggggttcag
      721 ggccacgtac aggcgcccgt ccgcctcgtc gctggcgtac tccgacagca gccgaaaccc
      781 ctgccgcttg cggccattct gggcgatgat ggataccttc caaaggcgct cgatgcagtc
      841 ctgtatgtgc ttgagcgccc caccactatc gacctctgcc ccgatttcct ttgccagcgc
      901 ccgatagcta cctttgacca ccatggcatc agcggtgacg gcctcccact tgggttccag
      961 gaacagccgg agctgccgtc cgccttcggt cttgggttcc gggccaagca ctaggccatt
     1021 aggcccagcc atggccacca gcccttgcag gatgcgcaga tcatcagcgc ccagcggctc
     1081 cgggccgctg aactcgatcc gcttgccgtc gccgtagtca tacgtcacgt ccagcttgct
     1141 gcgcttgcgc tcgccccgct tgagggcacg gaacaggccg ggggccagac agtgcgccgg
     1201 gtcgtgccgg acgtggctga ggctgtgctt gttcttaggc ttcaccacgg ggcaccccct
     1261 tgctcttgcg ctgcctctcc agcacggcgg gcttgagcac cccgccgtca tgccgcctga
     1321 accaccgatc agcgaacggt gcgccatagt tggccttgct cacaccgaag cggacgaaga
     1381 accggcgctg gtcgtcgtcc acaccccatt cctcggcctc ggcgctggtc atgctcgaca
     1441 ggtaggactg ccagcggatg ttatcgacca gtaccgagct gccccggctg gcctgctgct
     1501 ggtcgcctgc gcccatcatg gccgcgccct tgctggcatg gtgcaggaac acgatagagc
     1561 acccggtatc ggcggcgatg gcctccatgc gaccgatgac ctgggccatg gggccgctgg
     1621 cgttttcttc ctcgatgtgg aaccggcgca gcgtgtccag caccatcagg cggcggccct
     1681 cggcggcgcg cttgaggccg tcgaaccact ccggggccat gatgttgggc aggctgccga
     1741 tcagcggctg gatcagcagg ccgtcagcca cggcttgccg ttcctcggcg ctgaggtgcg
     1801 ccccaagggc gtgcaggcgg tgatgaatgg cggtgggcgg gtcttcggcg ggcaggtaga
     1861 tcaccgggcc ggtgggcagt tcgcccacct ccagcagatc cggcccgcct gcaatctgtg
     1921 cggccagttg cagggccagc atggatttac cggcaccacc gggcgacacc agcgccccga
     1981 ccgtaccggc caccatgttg ggcaaaacgt agtccagcgg tggcggcgct gctgcgaacg
     2041 cctccagaat attgataggc ttatgggtag ccattgattg cctcctttgc aggcagttgg
     2101 tggttaggcg ctggcggggt cactaccccc gccctgcgcc gctctgagtt cttccaggca
     2161 ctcgcgcagc gcctcgtatt cgtcgtcggt cagccagaac ttgcgctgac gcatcccttt
     2221 ggccttcatg cgctcggcat atcgcgcttg gcgtacagcg tcagggctgg ccagcaggtc
     2281 gccggtctgc ttgtcctttt ggtctttcat atcagtcacc gagaaacttg ccggggccga
     2341 aaggcttgtc ttcgcggaac aaggacaagg tgcagccgtc aaggttaagg ctggccatat
     2401 cagcgactga aaagcggcca gcctcggcct tgtttgacgt ataaccaaag ccaccgggca
     2461 accaatagcc cttgtcactt ttgatcaggt agaccgaccc tgaagcgctt ttttcgtatt
     2521 ccataaaacc cccttctgtg cgtgagtact catagtataa caggcgtgag taccaacgca
     2581 agcactacat gctgaaatct ggcccgcccc tgtccatgcc tcgctggcgg ggtgccggtg
     2641 cccgtgccag ctcggcccgc gcaagctgga cgctgggcag acccatgacc ttgctgacgg
     2701 tgcgctcgat gtaatccgct tcgtggccgg gcttgcgctc tgccagcgct gggctggcct
     2761 cggccatggc cttgccgatt tcctcggcac tgcggccccg gctggccagc ttctgcgcgg
     2821 cgataaagtc gcacttgctg aggtcatcgc cgaagcgctt gaccagcccg gccatctcgc
     2881 tgcggtactc gtccagcgcc gtgcgccggt ggcggctaag ctgccgctcg gacagttcga
     2941 ggctggccag cctgcgggcc ttctcctgct gccgctgggc ctgctcgatc tgctggccag
     3001 cctgctgcac cagcgccggg ccagcggtgg cggtcttgcc cttggattca cgcagcagca
     3061 cccacggctg ataaccggcg cgggtggtgt gcttgtcctt gcggttggtg aagcccgcca
     3121 agcggccata gtggcggctg tcggcgctgg ccgggtcggc gtcgtactcg ctggccagcg
     3181 tccgggcaat ctgtccccga agttcaccgc ctgcggcgtc ggccaccttg acccatgcct
     3241 gatagttctt cgggctggtt tccactacca gggcaggctc ccggccctcg gctttcatgt
     3301 catccaggtc aaactcgctg aggtcgtcca ccagcaccag accatgccgc tcctgctcgg
     3361 cgggcctgat atacacgtca ttgccctggg cattcatccg cttgagccat ggcgtgttct
     3421 ggagcacttc ggcggctgac cattcccggt tcatcatctg gccggtggtg gcgtccctga
     3481 cgccgatatc gaagcgctca cagcccatgg ccttgagctg tcggcctatg gcctgcaaag
     3541 tcctgtcgtt cttcatcggg ccaccaagcg cagccagatc gagccgtcct cggttgtcag
     3601 tggcgtcagg tcgagcaaga gcaacgatgc gatcagcagc accaccgtag gcatcatgga
     3661 agccagcatc acggttagcc atagcttcca gtgccacccc cgcgacgcgc tccgggcgct
     3721 ctgcgcggcg ctgctcacct cggcggctac ctcccgcaac tctttggcca gctccaccca
     3781 tgccgcccct gtctggcgct gggctttcag ccactccgcc gcctgcgcct cgctggcctg
     3841 ctgggtctgg ctcatgacct gccgggcttc gtcggccagt gtcgccatgc tctgggccag
     3901 cggttcgatc tgctccgcta actcgttgat gcctctggat ttcttcactc tgtcgattgc
     3961 gttcatggtc tattgcctcc cggtattcct gtaagtcgat gatctgggcg ttggcggtgt
     4021 cgatgttcag ggccacgtct gcccggtcgg tgcggatgcc ccggccttcc atctccacca
     4081 cgttcggccc caggtgaaca ccgggcaggc gctcgatgcc ctgcgcctca agtgttctgt
     4141 ggtcaatgcg ggcgtcgtgg cctgcccgct ctaatgcccg gttggcatgg tcggcccatg
     4201 cctcgcgggt ctgctcaagc catgccttgg gcttgagcgc ttcggtcttc tgtgccccgc
     4261 ccttctccgg ggtcttgccg ttgtaccgct tgaaccactg agcggcgggc cgctcgatgc
     4321 cgtcattgat ccgctcggag atcatcaggt ggcagtgcgg gttctcgccg ccaccggcat
     4381 ggatggccag cgtatacggc aggcgctcgg caccggtcag gtgctgggcg aactcggacg
     4441 ccagcgcctt ctgctggtcg agggtcagct cgaccggcag ggcaaattcg acctccttga
     4501 acagccgccc attggcgcgt tcatacaggt cggcagcatc ccagtagtcg gcgggccgct
     4561 cgacgaactc cggcatgtgc ccggattcgg cgtgcaagac ttcatccatg tcgcgggcat
     4621 acttgccttc gcgctggatg aagtcggcct tgccttcgcg ctggatgaag tcggccttgg
     4681 ccctggccga ttggccgccc gacctgctgc cggttttcgc cgtaaggtga taaatcgcca
     4741 tgctgcctcg ctgttgcttt tgcttttcgg ctccatgcaa tggccctcgg agagcgcacc
     4801 gcccgaaggg tggccgttag gccagtttct cgaagagaaa ccggtaagtg cgccctcccc
     4861 tacaaagtag ggtcgggatt gccgccgctg tgcctccatg atagcctacg agacagcaca
     4921 ttaacaatgg ggtgtcaaga tggttaaggg gagcaacaag gcggcggatc ggctggccaa
     4981 gctcgaagaa caacgagcgc gaatcaatgc cgaaattcag cgggtgcggg caagggaaca
     5041 gcagcaagag cgcaagaacg aaacaaggcg caaggtgctg gtgggggcca tgattttggc
     5101 caaggtgaac agcagcgagt ggccggagga tcggctcatg gcggcaatgg atgcgtacct
     5161 tgaacgcgac cacgaccgcg ccttgttcgg tctgccgcca cgccagaagg atgagccggg
     5221 ctgaatgatc gaccgagaca ggccctgcgg ggctgcacac gcgcccccac ccttcgggta
     5281 gggggaaagg ccgctaaagc ggctaaaagc gctccagcgt atttctgcgg ggtttggtgt
     5341 ggggtttagc gggctttgcc cgcctttccc cctgccgcgc agcggtgggg cggtgtgtag
     5401 cctagcgcag cgaatagacc agctatccgg cctctggccg ggcatattgg gcaagggcag
     5461 cagcgcccca caagggcgct gataaccgcg cctagtggat tattcttaga taatcatgga
     5521 tggatttttc caacaccccg ccagcccccg cccctgctgg gtttgcaggt ttgggggcgt
     5581 gacagttatt gcaggggttc gtgacagtta ttgcaggggg gcgtgacagt tattgcaggg
     5641 gttcgtgaca gttagtacgg gagtgacggg cactggctgg caatgtctag caacggcagg
     5701 catttcggct gagggtaaaa gaactttccg ctaagcgata gactgtatgt aaacacagta
     5761 ttgcaaggac gcggaacatg cctcatgtgg cggccctcgc gagaattaat tcagataaaa
     5821 aaaatcctta gctttcgcta aggatgattt ctagcgatga ccctgctgat tggttcgctg
     5881 accgctagca tcgcgccgcg cgcgcggatg ctagccaatg gcttattttc ctgctaattg
     5941 gtaataccat ggtaccgctc tgccagtgtt acaaccaatt aaccaattct gattagaaaa
     6001 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     6061 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     6121 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     6181 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     6241 gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac
     6301 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     6361 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     6421 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     6481 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     6541 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     6601 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     6661 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     6721 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctggagc
     6781 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     6841 acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt
     6901 gagacacaac gtggaccggt atcccgccgc cgccgccgga taccggtttc gaattgacaa
     6961 ttaatcatcg gctcgtataa tggtacccag atctccgcgg ccgccttgtt taactttaag
     7021 aaggagccct tcaccatgag taaaggagaa gaacttttca ctggagttgt cccaattctt
     7081 gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt
     7141 gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt
     7201 ccatggccaa cacttgtcac tactttcgcg tatggtcttc aatgctttgc gagataccca
     7261 gatcatatga aacagcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa
     7321 agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa
     7381 ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac
     7441 attcttggac acaaattgga atacaactat aactcacaca atgtatacat catggcagac
     7501 aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga agatggaagc
     7561 gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta
     7621 ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga
     7681 gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa
     7741 ctatacaaat aataaaaggg tgggcgcgcc gacccagctt tcttgtacaa actcggccct
     7801 gcaggagaag gccatcctga cggatggcct ttttgaagct ttctagaatc gggggccccc
     7861 ggggggat
//
