LOCUS       sequence-382435-        8996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression in A. baumannii for CRISPRi. Replicative plasmid, 
            KmR. Contains mrfp nontargeting control guide sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8996)
  AUTHORS   Bai J, Dai Y, Farinha A, Tang AY, Syal S, Vargas-Cuebas G, van 
            Opijnen T, Isberg RR, Geisinger E
  TITLE     Essential Gene Analysis in Acinetobacter baumannii by High-Density 
            Transposon Mutagenesis and CRISPR Interference.
  JOURNAL   J Bacteriol. 2021 May 20;203(12):e0056520. doi: 10.1128/JB.00565-20.
            Epub 2021 Mar 29.
  PUBMED    33782056
REFERENCE   2  (bases 1 to 8996)
  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: "J 
            Bacteriol."; date: "2021-05-20"; volume: "203(12):e0056520. doi"; 
            pages: " 10.1128/JB.00565-20. Epub 2021 Mar 29"
COMMENT     Sequence Label: pJE53
FEATURES             Location/Qualifiers
     source          1..8996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(95..683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(175..194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1217..2008)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="AKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKMT
                     DSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYEDE
                     QSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDTP
                     FKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIAF
                     CVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        join(2092..2097,2098..2114,2115..2120)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        2468..2502
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        2523..2598
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2619..2648
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(8657..8679)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    8765..8905
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     8820..8839
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(8920..8937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
       61 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
      241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
      301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
      361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
      421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
      481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
      541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
      601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
      661 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
      721 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
      781 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
      841 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
      901 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
      961 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1021 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1081 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1141 atagtttgcg caacgttgtt gccattgctg cagacagctt gtctgtaagc ggatgccggg
     1201 agcagacaag cccgtcctaa aacaattcat ccagtaaaat ataatatttt attttctccc
     1261 aatcaggctt gatccccagt aagtcaaaaa atagctcgac atactgttct tccccgatat
     1321 cctccctgat cgaccggacg cagaaggcaa tgtcatacca cttgtccgcc ctgccgcttc
     1381 tcccaagatc aataaagcca cttactttgc catctttcac aaagatgttg ctgtctccca
     1441 ggtcgccgtg ggaaaagaca agttcctctt cgggcttttc cgtctttaaa aaatcataca
     1501 gctcgcgcgg atctttaaat ggagtgtctt cttcccagtt ttcgcaatcc acatcggcca
     1561 gatcgttatt cagtaagtaa tccaattcgg ctaagcggct gtctaagcta ttcgtatagg
     1621 gacaatccga tatgtcgatg gagtgaaaga gcctgatgca ctccgcatac agctcgataa
     1681 tcttttcagg gctttgttca tcttcatact cttccgagca aaggacgcca tcggcctcac
     1741 tcatgagcag attgctccag ccatcatgcc gttcaaagtg caggaccttt ggaacaggca
     1801 gctttccttc cagccatagc atcatgtcct tttcccgttc cacatcatag gtggtccctt
     1861 tataccggct gtccgtcatt tttaaatata ggttttcatt ttctcccacc agcttatata
     1921 ccttagcagg agacattcct tccgtatctt ttacgcagcg gtatttttcg atcagttttt
     1981 tcaattccgg tgatattctc attttagcaa tttattattt ccttcctctt ttctacagta
     2041 tttaaagatt atatcacatt atccattaaa aatcaaaccg aattctcatg tttgacagct
     2101 tatcatcgat aagctttaat gcggtagttt atcacagtta aattgctaac gcagtcaggc
     2161 accgtgtatg aaatctaaca atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc
     2221 tgtaggcata ggcttggtta tgccggtact gccgggcctc ttgcgggata tcgtccattc
     2281 cgacagcatc gccagtcact atggcgtgct gctagcgcta tatgcgttga tgcaatttct
     2341 atgcgcaccc gttctcggag cactgtccga ccgctttggc cgccgcccag tcctgctcgc
     2401 ttcgctactt ggagccacta tcgactacgc gatcatggcg accacacccg tcctgtggat
     2461 ccgatctttg acagctagct cagtcctagg tataatacta gtaactttca gtttagcggt
     2521 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     2581 gtggcaccga gtcggtgctt tttttgaagc ttgggcccga acaaaaactc atctcagaag
     2641 aggatctgaa tagcgccgtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     2701 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     2761 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     2821 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     2881 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     2941 catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc
     3001 cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat
     3061 gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct
     3121 taccagccta acttcgatca ttggaccgct gatcgtcacg gcgatttatg ccgcctcggc
     3181 gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc
     3241 cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac
     3301 ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg
     3361 aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc
     3421 acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg
     3481 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     3541 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     3601 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     3661 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     3721 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3781 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3841 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3901 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3961 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     4021 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgctgc
     4081 tgttgctgct gttcaagtgt caattgctct actctgttgc tcgtctcaat gatagacata
     4141 tcaagccttt cagctttttt attgcgcttt ttgatttcat cattacgctt gacgatctcc
     4201 agttcctccc ccctgcgctt ggcgtgggtg gcttcaagtc cctccttgaa tgtcggctcg
     4261 atgtctagtc cacgatcctt atgactgcgg tgatcaactc tcacctcaag ccctgctcgc
     4321 tctaggtgga cgttcgtcag atccgccact ttttccctga tttttttcag cgttgagttc
     4381 tgatctagct ccctgacttt cttccctagc ccttgcggtg taaggcttct tgttgtcatt
     4441 agtatgtggg cgtgatggtt tctttcatca cttcccccat gcacatgagg ggcatggatc
     4501 gctacgtcca ccgccactcc ataggcttta actaaggact ggcacaactc ggtaacaagt
     4561 gcctgacgct gtgtcttatc cagttcatgc ggcaatgcta tctctacttc ctttgctaat
     4621 cttgcctcct gtttgatgtc accgttcttt tttagttcgg attgctctac ccgattccat
     4681 agggtttgac gatctaacat gtcaggactt gccccagttg gggcaaaaat ttgggtgtat
     4741 tcaatgcctg tttttttggt gtagtcctgc tcttttccgt acgtatcaca gtacaatttt
     4801 tcgcctgctc ggtatgctgc acatgccacg attgagcgac catctgacct cgaaatgttc
     4861 tgcatttcac aatggtaaat tgccattttc atcacctgtt tattaacagc cgccccaagt
     4921 tttgggcggt tggggtgtcg gggtttccct gaccgtactt gcaaaatttg gcgtagcaaa
     4981 aattttgcgt aagtgcgccc ttcgggaact ctgcgaggct aaaagcaaaa gcaagagcaa
     5041 taaggctttg actttgtttt ttgattttgc tcgctgcgct cggatcttga tgtaagtcga
     5101 gtttttgaag tagaacactt ttcacattga tgatggttca tacttcggaa aataggttga
     5161 gacacaggca ttaaaaatgg tcaaaaaaac agctatggaa ttgggtcaaa tgctcgatga
     5221 agaaaaggaa aaattggaac gtcaggaaaa ggcaagaaag gatctagctg acgctgtggt
     5281 gaaaggtcga gagcaaaaac aggcaaggtc agacgatgca aaacgcaaga tcctgatcgg
     5341 ggcttactta ctgaagaaac atgataatga catcaagaaa cttgtggaac aaaaccccga
     5401 ttttattggg tacattagag aaaatgataa gcacttattt tcataggcat tagtagttat
     5461 aaaaaaccct cataaatcga gggtttcttt tttatctgca ttactttttg aatcaatgta
     5521 cttttttagc ttgctatttt tcactgcata cagccgctta tactctttct caatcaatgc
     5581 ggtcaaaatg gctttttggc tctcccctgc tgcgcccacc atgtccgcaa gcatatccga
     5641 cacaccctta tctataaaca cctccaaacg ttgactatca aggcttttgc gtttttcacg
     5701 atatgctttt tgtcgttcgg cattggtttt tggcggctct tttagcatgt cctgtgtttt
     5761 ctgatcgttc gggtctttca tgtctttaac tcccatatag tatatcatag ttacaggata
     5821 acattgtttt tagttacagg ataacttttt ataaaattta tttgtaaaaa tttatgctat
     5881 cctgcaacta tttttaaatt acaggataac tatttaacgt taccctgtaa ctatagttat
     5941 cctgtaacgc taaattaatt accctgtaac atatattttg ttatcctgta acaactaaat
     6001 attgaaataa tactaccaaa gttaccctgt aacgaaaata actaggttaa aaaatgatcg
     6061 gattttaaca ttttgcgttg ttccaaaagt tatcaacagc ctagaacgtc ataggaagcg
     6121 attacagaca ctttatagct atcagcatgg gaacataagg gcaggatgaa atatgggtct
     6181 taaaacgcaa atggtgaggt tttagaggta ttttttgaag atgattaagg cggtttgttt
     6241 ttaaaatttt tggcggctct caggctgctt acattttaac cagttcagtg aaaagttctt
     6301 tttcagcaaa tttctgttta gcaccatagc taaaacttgc gtggaacata ttaagaattg
     6361 accgaaatga cacaatctca attatatttt ttttgaaaag ttttctttat caaatatttt
     6421 aaatcattga tttatatata agtatacatt cattttaata attaatcttt atttaacaat
     6481 gatttatcta tattcaattg tttaattatt cttactaata ttatctctat atcaatattt
     6541 tttatttaaa aacatatgtt tagtagtgct tttgattaaa gtaccagagg gagggagcag
     6601 agctgaatgg gaaatactca ccctagagcg attcttaaaa atcaccctaa agtattccca
     6661 ttcgatgtac cgtcggtcgg tcgctttcgc atcagggatg acatcactgt atcaagctgc
     6721 cactgttatg attacgattg atagcaccgc ctgaacacgc tcataaccgc caattaaatg
     6781 actactcatt gcgtccgcta ctctgttcag ttcccctagt aatagcgttt ttccgatgtg
     6841 tgctagcgtc actgtacctc atcacccaca catggacaga ttgagttaca gacattggct
     6901 aaatttttgg ggtctatgct tgacaaagcg agttaaaacc ttagaattta agacaggtac
     6961 attaagcccc tgtggtgaaa tcattagcgg tcgtcaaacc ttaatgcttt cgattgccga
     7021 tatgtcagta tcgaagatct gtaccccata aattacggta aagccccaag caattgcaag
     7081 gggcttttat cttttttaac aaaaaaaatt tataaatcag gattttataa cactaaataa
     7141 tccaaagtac atgagtaagt catgaccact cctgcgatgt gtgtagcact ctgagtatcc
     7201 gtattatatc agtgatgtca tatacaacca cataatgcgg atgtatcact aactccctag
     7261 tatctttctg tctgcctgtg cgccccatca gtcgattatc aacaagtaaa tctgtctttt
     7321 cttcaattaa atcatcaatt tcaatagctg ctatagggtt gcgttcttca agataatcat
     7381 agatatttct acgatcttgg cgagctgttt cagtccactc aatcttcatt cttcgctgcc
     7441 agtttcgcca atgttgcagc acgtctagcg gcaaactctg cctttacgtc ctcatgtgga
     7501 atcaacttgc ctgcattggc ttcatttaag ccgatctgta cctgctgctt gaaccatttg
     7561 tcatagtctg cggcttcctg ttgcttctta acaaagtcac gcatgaagtc acggattagc
     7621 tgcgcccccg accgatcaac agtcttagca aggcttgaaa attcttgctt taggtcatgg
     7681 tctacccgaa aggtaaaagt cgcttctgtc atgtttttca ctccaatgtg ttacatttga
     7741 atgctattgt aacacattaa aaagttaggt cattgtcttt tttcggctta ggctgttctt
     7801 ggtcataacg tggtttaaat gatggctgtc gctcattctc aagtaaaatt ttccgtttat
     7861 taacagcctg ttgcatctgc tcatagtaag gcatcaactg cggtaaaata tcagcaagtg
     7921 cctgtctgtc tttatccagg atacggctca attgcccgat ttttttcaag tcacgctcat
     7981 aggctattct gtcgctgatc tgctcgtatt tttgctttga acaccttatg tactcttgca
     8041 tatcgttact tgcccatgcc tgcgcctgaa gacgtgtaaa catggaaatg tctgaagcaa
     8101 catggtcgct gatatggtca tactcagcat ctttgacata gccgtatttt gataaatatc
     8161 tttcatagtc ctgccgtttc tttttgtcat cttcctgttt ttgtttagtt tctctttcta
     8221 tctgctgttg ttttcgttgc tgttcctgtt gctcctcttt tatctctctg tgataaatgg
     8281 aaaggctaga ttgatcatta tcccaacgtg tgagcatgtc ctcaaagccg taataatcag
     8341 cactaaacca acctgttttc actttcactg gtggcggcag tggctctccg atctccctta
     8401 gcagtgctgt agctgctcgt ccctctttga tcactttatc aagctgcaag cggtcgatat
     8461 tgtcttgcaa agccttgtaa aatctatgct ctgctcgctt ctcatccgtt tttacttcaa
     8521 tcaaatacac atctataagc gtttggcggt gttgttggtg gtcgtatagt gcctgttgtg
     8581 cggtggcata gactgcgggg gcttggtcta cggcatcctc cagctgcctc gcgcgtttcg
     8641 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     8701 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     8761 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     8821 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     8881 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     8941 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggt
//
