LOCUS       sequence-467189-        5509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Guide RNA production in Caulobacter crescentus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5509)
  AUTHORS   Filsinger GT, Mychack A, Lyerly E, Henriksen C, Bartlett TM, 
            Kuchwara H, Eitzinger S, Bernhardt TG, Walker S, Church GM, Wannier 
            TM
  TITLE     A diverse single-stranded DNA-annealing protein library enables 
            efficient genome editing across bacterial phyla.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Apr 29;122(17):e2414342122. doi: 
            10.1073/pnas.2414342122. Epub 2025 Apr 21.
  PUBMED    40258142
REFERENCE   2  (bases 1 to 5509)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-04-29"; volume: "122(17):e2414342122. 
            doi"; pages: " 10.1073/pnas.2414342122. Epub 2025 Apr 21"
COMMENT     Sequence Label: pORTMAGE-Cc2
FEATURES             Location/Qualifiers
     source          1..5509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             458..1252
                     /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(512..531)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1122..1141
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1600..1646
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2068..2111
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2254..2270)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2254..2270)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2302..2321)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2303..2321)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2331..2348)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2331..2347)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2340..2362)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(3055..3717)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      3718..4487
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc
       61 aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg
      121 aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg
      181 aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag
      241 cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt
      301 tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa
      361 gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc
      421 aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
      481 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
      541 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
      601 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
      661 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
      721 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
      781 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
      841 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
      901 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
      961 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     1021 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     1081 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     1141 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     1201 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     1261 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     1321 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     1381 atcctccagc gcggggatct catgctggag ttcttcgccc acccccatgg gcaaatatta
     1441 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     1501 tggcttccat gtcggcagaa tgcttaatga attacaacag tttttatata agctttcagt
     1561 cggcgcgaat gctccacgcc gcgcccagcg gcgcggatcc aaataaaacg aaaggctcag
     1621 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgc tgcagccagc cgtggtcggg
     1681 cagcaggtag aaggcgccct cgtcctgatc ctcgcccgaa acctccagcc cccggtcgat
     1741 ggcttcgacg acatagccgg ccgcgcggca ggtgtcggtg agcgcggcca gcagggcggc
     1801 ttcctggtca ggggtcaggt cggtcatggg caagaggtcc aggtcgtggt ttgtcggcgg
     1861 cttctagcat ggaccgcccg cgcccgtgag gccgaggatt tcgcgctggt cagacaacct
     1921 acttgccgtc cccacatgtt agcgctacca agtgccgagg gtcttcgaga agacctgttt
     1981 tagagctgtg aaaacagcga gttaaaataa ggcttagtcc gtactcaact tgaaaaggtg
     2041 gcaccgattc ggtgtttttt ttttttgata aaacgaaagg cccagtcttt cgactgagcc
     2101 tttcgtttta taaacgggtc ttgaggggtt ttttaagctt ctgcagctgg atccatggat
     2161 atctagattt ccgcaaaata aaaaagggga cctctagggt ccccaattaa ttagctaaag
     2221 ccgctagccc atgggaattc ctgcagcccg ggggatccac tagttctaga gcggccgcca
     2281 ccgcggtgga gctccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     2341 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2401 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2461 cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg
     2521 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgtactg cgatgagtgg
     2581 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggtgctacg
     2641 cctgaataag tgataataag cggatgaatg gcagaaattc gaaagcaaat tcgacccggt
     2701 cgtcggttca gggcagggtc gttaaatagc cgcttatgtc tattgctggt ttaccggttt
     2761 attgactacc ggaagcagtg tgaccgtgtg cttctcaaat gcctgaggcc agtttgctca
     2821 ggctctcccc gtggaggtaa taattgacga tatgatcatt tattctgcct cccagagcct
     2881 gataaaaacg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc
     2941 cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgaggcgg
     3001 ctacagccga tagtctggaa cagcgcactt acgggttgct gcgcaaccca agtgctaccg
     3061 gcgcggcagc gtgacccgtg tcggcggctc caacggctcg ccatcgtcca gaaaacacgg
     3121 ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc cattcctccg tttcggtcaa
     3181 ggctggcagg tctggttcca tgcccggaat gccgggctgg ctgggcggct cctcgccggg
     3241 gccggtcggt agttgctgct cgcccggata cagggtcggg atgcggcgca ggtcgccatg
     3301 ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc acggcggcac tgaacaccga
     3361 caggcgcaac tggtcgcggg gctggcccca cgccacgcgg tcattgacca cgtaggccga
     3421 cacggtgccg gggccgttga gcttcacgac ggagatccag cgctcggcca ccaagtcctt
     3481 gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc ttggaaagtg ttttctggct
     3541 gaccaccacg gcgttctggt ggcccatctg cgccacgagg tgatgcagca gcattgccgc
     3601 cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc gctttgcgtt ccgtttgcac
     3661 ccagtgaccg ggcttgttct tggcttgaat gccgatttct ctggactgcg tggccatgct
     3721 tatctccatg cggtagggtg ccgcacggtt gcggcaccat gcgcaatcag ctgcaacttt
     3781 tcggcagcgc gacaacaatt atgcgttgcg taaaagtggc agtcaattac agattttctt
     3841 taacctacgc aatgagctat tgcggggggt gccgcaatga gctgttgcgt accccccttt
     3901 tttaagttgt tgatttttaa gtctttcgca tttcgcccta tatctagttc tttggtgccc
     3961 aaagaagggc acccctgcgg ggttccccca cgccttcggc gcggctcccc ctccggcaaa
     4021 aagtggcccc tccggggctt gttgatcgac tgcgcggcct tcggccttgc ccaaggtggc
     4081 gctgccccct tggaaccccc gcactcgccg ccgtgaggct cggggggcag gcgggcgggc
     4141 ttcgccttcg actgccccca ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa
     4201 gccgctgcgc ggtcgctgcg cgagccttga cccgccttcc acttggtgtc caaccggcaa
     4261 gcgaagcgcg caggccgcag gccggaggct tttccccaga gaaaattaaa aaaattgatg
     4321 gggcaaggcc gcaggccgcg cagttggagc cggtgggtat gtggtcgaag gctgggtagc
     4381 cggtgggcaa tccctgtggt caagctcgtg ggcaggcgca gcctgtccat cagcttgtcc
     4441 agcagggttg tccacgggcc gagcgaagcg agccagccgg tggccgctcg cggccatcgt
     4501 ccacatatcc acgggctggc aagggagcgc agcgaccgcg cagggcgaag cccggagagc
     4561 aagcccgtag ggcgccgcag ccgccgtagg cggtcacgac tttgcgaagc aaagtctagt
     4621 gagtatactc aagcattgag tggcccgccg gaggcaccgc cttgcgctgc ccccgtcgag
     4681 ccggttggac accaaaaggg aggggcaggc atggcggcat acgcgatcat gcgatgcaag
     4741 aagctggcga aaatgggcaa cgtggcggcc agtctcaagc acgcctaccg cgagcgcgag
     4801 acgcccaacg ctgacgccag caggacgcca gagaacgagc actgggcggc cagcagcacc
     4861 gatgaagcga tgggccgact gcgcgagttg ctgccagaga agcggcgcaa ggacgctgtg
     4921 ttggcggtcg agtacgtcat gacggccagc ccggaatggt ggaagtcggc cagccaagaa
     4981 cagcaggcgg cgttcttcga gaaggcgcac aagtggctgg cggacaagta cggggcggat
     5041 cgcatcgtga cggccagcat ccaccgtgac gaaaccagcc cgcacatgac cgcgttcgtg
     5101 gtgccgctga cgcaggacgg caggctgtcg gccaaggagt tcatcggcaa caaagcgcag
     5161 atgacccgcg accagaccac gtttgcggcc gctgtggccg atctagggct gcaacggggc
     5221 atcgagggca gcaaggcacg tcacacgcgc attcaggcgt tctacgaggc cctggagcgg
     5281 ccaccagtgg gccacgtcac catcagcccg caagcggtcg agccacgcgc ctatgcaccg
     5341 cagggattgg ccgaaaagct gggaatctca aagcgcgttg agacgccgga agccgtggcc
     5401 gaccggctga caaaagcggt tcggcagggg tatgagcctg ccctacaggc cgccgcagga
     5461 gcgcgtgaga tgcgcaagaa ggccgatcaa gcccaagaga cggcccgag
//
