LOCUS       sequence-495291-        9889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FSD expressing SIBR-Cas system with intron 4, contains restriction 
            sites to introduce HR and spacer sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9889)
  TITLE     Vectors for KO generation in Shewanella glacialimarina
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9889)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pFSD-SIBR004
FEATURES             Location/Qualifiers
     source          1..9889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             11..982
                     /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
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     CDS             1493..2332
                     /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             2319..3170
                     /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"
     primer_bind     3419..3435
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3420..3436
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3470..3486)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3470..3486)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3817..4611
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3871..3890)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4481..4500
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        join(4660..4665,4666..4683,4684..4690)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     promoter        join(9025..9030,9031..9048,9049..9055)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     rep_origin      9368..9762
                     /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)"
ORIGIN
        1 gcgaggcagc atgaagaacg acaggacttt gcaggccata ggccgacagc tcaaggccat
       61 gggctgtgag cgcttcgata tcggcgtcag ggacgccacc accggccaga tgatgaaccg
      121 ggaatggtca gccgccgaag tgctccagaa cacgccatgg ctcaagcgga tgaatgccca
      181 gggcaatgac gtgtatatca ggcccgccga gcaggagcgg catggtctgg tgctggtgga
      241 cgacctcagc gagtttgacc tggatgacat gaaagccgag ggccgggagc ctgccctggt
      301 agtggaaacc agcccgaaga actatcaggc atgggtcaag gtggccgacg ccgcaggcgg
      361 tgaacttcgg gggcagattg cccggacgct ggccagcgag tacgacgccg acccggccag
      421 cgccgacagc cgccactatg gccgcttggc gggcttcacc aaccgcaagg acaagcacac
      481 cacccgcgcc ggttatcagc cgtgggtgct gctgcgtgaa tccaagggca agaccgccac
      541 cgctggcccg gcgctggtgc agcaggctgg ccagcagatc gagcaggccc agcggcagca
      601 ggagaaggcc cgcaggctgg ccagcctcga actgcccgag cggcagctta gccgccaccg
      661 gcgcacggcg ctggacgagt accgcagcga gatggccggg ctggtcaagc gcttcggtga
      721 tgacctcagc aagtgcgact ttatcgccgc gcagaagctg gccagccggg gccgcagtgc
      781 cgaggaaatc ggcaaggcca tggccgaggc cagcccagcg ctggcagagc gcaagcccgg
      841 ccacgaagcg gattacatcg agcgcaccgt cagcaaggtc atgggtctgc ccagcgtcca
      901 gcttgcgcgg gccgagctgg cacgggcacc ggcaccccgc cagcgaggca tggacagggg
      961 cgggccagat ttcagcatgt agtgcttgcg ttggtactca cgcctgttat actatgagta
     1021 ctcacgcaca gaagggggtt ttatggaata cgaaaaaagc gcttcagggt cggtctacct
     1081 gatcaaaagt gacaagggct attggttgcc cggtggcttt ggttatacgt caaacaaggc
     1141 cgaggctggc cgcttttcag tcgctgatat ggccagcctt aaccttgacg gctgcacctt
     1201 gtccttgttc cgcgaagaca agcctttcgg ccccggcaag tttctcggtg actgatatga
     1261 aagaccaaaa ggacaagcag accggcgacc tgctggccag ccctgacgct gtacgccaag
     1321 cgcgatatgc cgagcgcatg aaggccaaag ggatgcgtca gcgcaagttc tggctgaccg
     1381 acgacgaata cgaggcgctg cgcgagtgcc tggaagaact cagagcggcg cagggcgggg
     1441 gtagtgaccc cgccagcgcc taaccaccaa ctgcctgcaa aggaggcaat caatggctac
     1501 ccataagcct atcaatattc tggaggcgtt cgcagcagcg ccgccaccgc tggactacgt
     1561 tttgcccaac atggtggccg gtacggtcgg ggcgctggtg tcgcccggtg gtgccggtaa
     1621 atccatgctg gccctgcaac tggccgcaca gattgcaggc gggccggatc tgctggaggt
     1681 gggcgaactg cccaccggcc cggtgatcta cctgcccgcc gaagacccgc ccaccgccat
     1741 tcatcaccgc ctgcacgccc ttggggcgca cctcagcgcc gaggaacggc aagccgtggc
     1801 tgacggcctg ctgatccagc cgctgatcgg cagcctgccc aacatcatgg ccccggagtg
     1861 gttcgacggc ctcaagcgcg ccgccgaggg ccgccgcctg atggtgctgg acacgctgcg
     1921 ccggttccac atcgaggaag aaaacgccag cggccccatg gcccaggtca tcggtcgcat
     1981 ggaggccatc gccgccgata ccgggtgctc tatcgtgttc ctgcaccatg ccagcaaggg
     2041 cgcggccatg atgggcgcag gcgaccagca gcaggccagc cggggcagct cggtactggt
     2101 cgataacatc cgctggcagt cctacctgtc gagcatgacc agcgccgagg ccgaggaatg
     2161 gggtgtggac gacgaccagc gccggttctt cgtccgcttc ggtgtgagca aggccaacta
     2221 tggcgcaccg ttcgctgatc ggtggttcag gcggcatgac ggcggggtgc tcaagcccgc
     2281 cgtgctggag aggcagcgca agagcaaggg ggtgccccgt ggtgaagcct aagaacaagc
     2341 acagcctcag ccacgtccgg cacgacccgg cgcactgtct ggcccccggc ctgttccgtg
     2401 ccctcaagcg gggcgagcgc aagcgcagca agctggacgt gacgtatgac tacggcgacg
     2461 gcaagcggat cgagttcagc ggcccggagc cgctgggcgc tgatgatctg cgcatcctgc
     2521 aagggctggt ggccatggct gggcctaatg gcctagtgct tggcccggaa cccaagaccg
     2581 aaggcggacg gcagctccgg ctgttcctgg aacccaagtg ggaggccgtc accgctgatg
     2641 ccatggtggt caaaggtagc tatcgggcgc tggcaaagga aatcggggca gaggtcgata
     2701 gtggtggggc gctcaagcac atacaggact gcatcgagcg cctttggaag gtatccatca
     2761 tcgcccagaa tggccgcaag cggcaggggt ttcggctgct gtcggagtac gccagcgacg
     2821 aggcggacgg gcgcctgtac gtggccctga accccttgat cgcgcaggcc gtcatgggtg
     2881 gcggccagca tgtgcgcatc agcatggacg aggtgcgggc gctggacagc gaaaccgccc
     2941 gcctgctgca ccagcggctg tgtggctgga tcgaccccgg caaaaccggc aaggcttcca
     3001 tagatacctt gtgcggctat gtctggccgt cagaggccag tggttcgacc atgcgcaagc
     3061 gccgccagcg ggtgcgcgag gcgttgccgg agctggtcgc gctgggctgg acggtaaccg
     3121 agttcgcggc gggcaagtac gacatcaccc ggcccaaggc ggcaggctga ccccccccac
     3181 tctattgtaa acaagacatt tttatctttt atattcaatg gcttattttc ctgctaattg
     3241 gtaataccat gaaaaatacc atgctcagaa aaggcttaac aatattttga aaaattgcct
     3301 actgagcgct gccgcacagc tccataggcc gctttcctgg ctttgcttcc agatgtatgc
     3361 tctatttact atagggcgaa ttgggtaccg gccggcctta attaagatct ggcgcgcctc
     3421 gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg atccactagt
     3481 tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttaaa atcgcagaaa
     3541 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     3601 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     3661 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     3721 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     3781 agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3841 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3901 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3961 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4021 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4081 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4141 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4201 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4261 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4321 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4381 ggcgatgcct gcttgccaaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4441 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4501 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4561 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4621 tggggtcaat tgcggataac aatttcacac aggaggccgt ttacacttta tgcttccggc
     4681 tcgtataatg tgtggggaaa ccacaacggt ttccctctag aaataatttt gtttaactat
     4741 aagaaggaga tatacatatg tcctctggtt aattgaggcc tgagtataag gtgacttata
     4801 cttgtaatct atctaaacgg ggaacctctc tagtagacaa tcccgtgcta aattgatacc
     4861 agcatcgtct tgatgccctt ggcagcataa atgcctaacg actatccctt tggggagtag
     4921 ggtcaagtga ctcgaaacga tagacaactt gctttaacaa gttggagata tagtctgctc
     4981 tgcatggtga catgcagctg gatataattc cggggtaaga ttaacgacct tatctgaaca
     5041 taatgctatc aatttatcaa gaatttgtta ataaatatag tttaagtaaa actctaagat
     5101 ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt ttgattttag
     5161 atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat aaatatcatc
     5221 agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta ttacaaaact
     5281 attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa aaagatttta
     5341 aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac tcagagaaat
     5401 ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag tcagatttaa
     5461 ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc aatagtgata
     5521 tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg acaacttatt
     5581 ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt cctacatcta
     5641 ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa gctaagtatg
     5701 agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa aaagatttgg
     5761 cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa agagtttttt
     5821 cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa agtggtatta
     5881 ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca aagagaaaag
     5941 gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca ctcaaaaaat
     6001 ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa tcttttgtaa
     6061 ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt tatgagcaaa
     6121 tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct ttattatttg
     6181 atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat gataaatctc
     6241 ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca gcggtactag
     6301 aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag aaagagcaag
     6361 aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact ataaagcttg
     6421 ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt gaagaaatac
     6481 ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac aaagacaatt
     6541 tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt caagctagtg
     6601 cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat ctcttacata
     6661 aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta gacaaggatg
     6721 agcattttta tctagtattt gaggagtgct actttgagct agcgaatata gtgcctcttt
     6781 ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa tttaagctca
     6841 attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct gacaatacgg
     6901 caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag aaaaataaca
     6961 aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa aaaattgttt
     7021 ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct gctaaatcta
     7081 taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc acacatacaa
     7141 aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa gattgccgaa
     7201 aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa gattttggat
     7261 ttagattttc tgatactcaa agatataatt ctatagatga attttataga gaagttgaaa
     7321 atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat agcgtagtta
     7381 atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct tatagcaaag
     7441 ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga aatcttcaag
     7501 atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa tcaataccta
     7561 aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat aatcctaaaa
     7621 aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa gataagtttt
     7681 tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag tttaatgatg
     7741 aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt atagatagag
     7801 gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc atcaaacaag
     7861 atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat aagcttgctg
     7921 caatagagaa agatagggat tcagctagga aagactggaa aaagataaat aacatcaaag
     7981 agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta gttatagagt
     8041 ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg cgtttcaagg
     8101 tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta aactatctag
     8161 ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat cagctaacag
     8221 caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac tatgtaccag
     8281 ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta tatcctaagt
     8341 atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt tgttataacc
     8401 ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac aaggctgcca
     8461 aaggcaagtg gactatagct agctttggga gtagattgat taactttaga aattcagata
     8521 aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag aaattgctaa
     8581 aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt tgcggtgaga
     8641 gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta caaatgcgta
     8701 actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat gtaaatggca
     8761 atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat gccaatggtg
     8821 cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat aatcaagagg
     8881 gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg cagaatagga
     8941 ataactaagc ggccgcataa tgatgtgtta tcattgatgc gaggtcgcct atacctcccc
     9001 gcttcggcgg ggttttttcc cgggtttaca ctttatgctt ccggctcgta taatgtgtgg
     9061 tctaagaact ttaaataatt tctactgttg tagatagaga cccctgagtg acgagcaacg
     9121 agactggggt ctctgtctaa gaactttaaa taatttctac tgttgtagat agataccgga
     9181 caacgtgtct ccccgcttcg gcggggtttt ttcctaggtt gtgtctcaaa atctctgcaa
     9241 ttgtccttgc aatactgtgt ttacatacag tctatcgctt agcggaaagt tcttttaccc
     9301 tcagccgaaa tgcctgccgt tgctagacat tgccagccag tgcccgtcac tcccgtacta
     9361 actgtcacga acccctgcaa taactgtcac gcccccctgc aataactgtc acgaacccct
     9421 gcaataactg tcacgccccc aaacctgcaa acccagcagg ggcgggggct ggcggggtgt
     9481 tggaaaaatc catccatgat tatctaagaa taatccacta ggcgcggtta tcagcgccct
     9541 tgtggggcgc tgctgccctt gcccaatatg cccggccaga ggccggatag ctggtctatt
     9601 cgctgcgcta ggctacacac cgccccaccg ctgcgcggca gggggaaagg cgggcaaagc
     9661 ccgctaaacc ccacaccaaa ccccgcagaa atacgctgga gcgcttttag ccgctttagc
     9721 ggcctttccc cctacccgaa gggtgggggc gcgtgtgcag ccccgcaggg cctgtctcgg
     9781 tcgatcattc agcgttgctc cccttaacca tcttgacacc ccattgttaa tgtgctgtct
     9841 cgtaggctat catggaggca cagcatggag ccgaaaagca aaagcaaca
//
