LOCUS       sequence-434129-        6073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression for S. stutzeri nifL deletion on an RSF1010 
            backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6073)
  AUTHORS   Venkataraman M, Ynigez-Gutierrez A, Infante V, MacIntyre A, 
            Fernandes-Junior PI, Ane JM, Pfleger B
  TITLE     Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.
  JOURNAL   ACS Synth Biol. 2023 Dec 15;12(12):3623-3634. doi: 
            10.1021/acssynbio.3c00414. Epub 2023 Nov 21.
  PUBMED    37988619
REFERENCE   2  (bases 1 to 6073)
  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"; doi: 
            "10.1021/acssynbio.3c00414"; journalName: "ACS Synth Biol"; date: 
            "2023-12-15- 15"; volume: "12"; issue: "12"; pages: "3623-3634"
COMMENT     Sequence Label: pMVV207
FEATURES             Location/Qualifiers
     source          1..6073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        71..146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             167..196
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             212..229
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(285..302)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      455..501
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(554..571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(706..1557)
                     /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(1544..2383)
                     /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(2894..3865)
                     /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"
     rep_origin      3972..4366
                     /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(4389..4483)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4656..5846)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTITLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILQALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     5475..5494
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(5894..5899,5900..5916,5917..5922))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     6006..6024
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(6028..6071)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt gcggttggca
       61 tagaggatgt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      121 ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac aaaaactcat
      181 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac
      241 ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
      301 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
      361 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
      421 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
      481 agactgggcc tttcgtttta tctgttgttt gtcggtgaac tggatcctta ctcgagtcta
      541 gactgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      601 tatcagctca ctcaaaggcg gtaatacggc ctttctgcgt ttatacctag gctacagccg
      661 atagtctgga acagcgcact tacgggttgc tgcgcaaccc aagtgtcagc ctgccgcctt
      721 gggccgggtg atgtcgtact tgcccgccgc gaactcggtt accgtccagc ccagcgcgac
      781 cagctccggc aacgcctcgc gcacccgctg gcggcgcttg cgcatggtcg aaccactggc
      841 ctctgacggc cagacatagc cgcacaaggt atctatggaa gccttgccgg ttttgccggg
      901 gtcgatccag ccacacagcc gctggtgcag caggcgggcg gtttcgctgt ccagcgcccg
      961 cacctcgtcc atgctgatgc gcacatgctg gccgccaccc atgacggcct gcgcgatcaa
     1021 ggggttcagg gccacgtaca ggcgcccgtc cgcctcgtcg ctggcgtact ccgacagcag
     1081 ccgaaacccc tgccgcttgc ggccattctg ggcgatgatg gataccttcc aaaggcgctc
     1141 gatgcagtcc tgtatgtgct tgagcgcccc accactatcg acctctgccc cgatttcctt
     1201 tgccagcgcc cgatagctac ctttgaccac catggcatca gcggtgacgg cctcccactt
     1261 gggttccagg aacagccgga gctgccgtcc gccttcggtc ttgggttccg ggccaagcac
     1321 taggccatta ggcccagcca tggccaccag cccttgcagg atgcgcagat catcagcgcc
     1381 cagcggctcc gggccgctga actcgatccg cttgccgtcg ccgtagtcat acgtcacgtc
     1441 cagcttgctg cgcttgcgct cgccccgctt gagggcacgg aacaggccgg gggccagaca
     1501 gtgcgccggg tcgtgccgga cgtggctgag gctgtgcttg ttcttaggct tcaccacggg
     1561 gcaccccctt gctcttgcgc tgcctctcca gcacggcggg cttgagcacc ccgccgtcat
     1621 gccgcctgaa ccaccgatca gcgaacggtg cgccatagtt ggccttgctc acaccgaagc
     1681 ggacgaagaa ccggcgctgg tcgtcgtcca caccccattc ctcggcctcg gcgctggtca
     1741 tgctcgacag gtaggactgc cagcggatgt tatcgaccag taccgagctg ccccggctgg
     1801 cctgctgctg gtcgcctgcg cccatcatgg ccgcgccctt gctggcatgg tgcaggaaca
     1861 cgatagagca cccggtatcg gcggcgatgg cctccatgcg accgatgacc tgggccatgg
     1921 ggccgctggc gttttcttcc tcgatgtgga accggcgcag cgtgtccagc accatcaggc
     1981 ggcggccctc ggcggcgcgc ttgaggccgt cgaaccactc cggggccatg atgttgggca
     2041 ggctgccgat cagcggctgg atcagcaggc cgtcagccac ggcttgccgt tcctcggcgc
     2101 tgaggtgcgc cccaagggcg tgcaggcggt gatgaatggc ggtgggcggg tcttcggcgg
     2161 gcaggtagat caccgggccg gtgggcagtt cgcccacctc cagcagatcc ggcccgcctg
     2221 caatctgtgc ggccagttgc agggccagca tggatttacc ggcaccaccg ggcgacacca
     2281 gcgccccgac cgtaccggcc accatgttgg gcaaaacgta gtccagcggt ggcggcgctg
     2341 ctgcgaacgc ctccagaata ttgataggct tatgggtagc cattgattgc ctcctttgca
     2401 ggcagttggt ggttaggcgc tggcggggtc actacccccg ccctgcgccg ctctgagttc
     2461 ttccaggcac tcgcgcagcg cctcgtattc gtcgtcggtc agccagaact tgcgctgacg
     2521 catccctttg gccttcatgc gctcggcata tcgcgcttgg cgtacagcgt cagggctggc
     2581 cagcaggtcg ccggtctgct tgtccttttg gtctttcata tcagtcaccg agaaacttgc
     2641 cggggccgaa aggcttgtct tcgcggaaca aggacaaggt gcagccgtca aggttaaggc
     2701 tggccatatc agcgactgaa aagcggccag cctcggcctt gtttgacgta taaccaaagc
     2761 caccgggcaa ccaatagccc ttgtcacttt tgatcaggta gaccgaccct gaagcgcttt
     2821 tttcgtattc cataaaaccc ccttctgtgc gtgagtactc atagtataac aggcgtgagt
     2881 accaacgcaa gcactacatg ctgaaatctg gcccgcccct gtccatgcct cgctggcggg
     2941 gtgccggtgc ccgtgccagc tcggcccgcg caagctggac gctgggcaga cccatgacct
     3001 tgctgacggt gcgctcgatg taatccgctt cgtggccggg cttgcgctct gccagcgctg
     3061 ggctggcctc ggccatggcc ttgccgattt cctcggcact gcggccccgg ctggccagct
     3121 tctgcgcggc gataaagtcg cacttgctga ggtcatcacc gaagcgcttg accagcccgg
     3181 ccatctcgct gcggtactcg tccagcgccg tgcgccggtg gcggctaagc tgccgctcgg
     3241 gcagttcgag gctggccagc ctgcgggcct tctcctgctg ccgctgggcc tgctcgatct
     3301 gctggccagc ctgctgcacc agcgccgggc cagcggtggc ggtcttgccc ttggattcac
     3361 gcagcagcac ccacggctga taaccggcgc gggtggtgtg cttgtccttg cggttggtga
     3421 agcccgccaa gcggccatag tggcggctgt cggcgctggc cgggtcggcg tcgtactcgc
     3481 tggccagcgt ccgggcaatc tgcccccgaa gttcaccgcc tgcggcgtcg gccaccttga
     3541 cccatgcctg atagttcttc gggctggttt ccactaccag ggcaggctcc cggccctcgg
     3601 ctttcatgtc atccaggtca aactcgctga ggtcgtccac cagcaccaga ccatgccgct
     3661 cctgctcggc gggcctgata tacacgtcat tgccctgggc attcatccgc ttgagccatg
     3721 gcgtgttctg gagcacttcg gcggctgacc attcccggtt catcatctgg ccggtggtgg
     3781 cgtccctgac gccgatatcg aagcgctcac agcccatggc cttgagctgt cggcctatgg
     3841 cctgcaaagt cctgtcgttc ttcatcgggc caccaagcga ttcccacaca ttatacgagc
     3901 cggaagcata aagtgtagct agatccgaag gatgagccgg gctgaatgat cgaccgagac
     3961 aggccctgcg gggctgcaca cgcgccccca cccttcgggt agggggaaag gccgctaaag
     4021 cggctaaaag cgctccagcg tatttctgcg gggtttggtg tggggtttag cgggctttgc
     4081 ccgcctttcc ccctgccgcg cagcggtggg gcggtgtgta gcctagcgca gcgaatagac
     4141 cagctatccg gcctctggcc gggcatattg ggcaagggca gcagcgcccc acaagggcgc
     4201 tgataaccgc gcctagtgga ttattcttag ataatcatgg atggattttt ccaacacccc
     4261 gccagccccc gcccctgctg ggtttgcagg tttgggggcg tgacagttat tgcaggggtt
     4321 cgtgacagtt attgcagggg ggcgtgacag ttattgcagg ggttcgtgac agttagacta
     4381 gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc
     4441 agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga gctctcgaac
     4501 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     4561 cgggagcggc gataccgtaa agcacgagga aagtgacagg cacattatgc atcttggagt
     4621 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     4681 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     4741 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
     4801 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     4861 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     4921 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     4981 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     5041 ggaccagtga cgaaggcttg agcgagggct tgcaagattc cgaataccgc aagcgacagg
     5101 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     5161 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     5221 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5281 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5341 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5401 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5461 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     5521 gtgatgccgg ccacgatgcg tccggcgtag aggatccaca ggacgggtgt ggtcgccatg
     5581 atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag
     5641 cggtcggaca gtgctccgag aacgggtgcg cataggaatt gcatcaacgc atatagcgct
     5701 agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc
     5761 ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgatggt gccgaggatg
     5821 acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt
     5881 gataaactac cgcattaaag cttatcgatg ataagctgtc aaacataggg gttccgcgca
     5941 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6001 ataaaaatag gcgtatcacg aggcagaatt tcagataaaa aaaatcctta gctttcgcta
     6061 aggatgattt ctg
//
