LOCUS       sequence-497877-        8836 bp    DNA     circular SYN 21-JAN-2026
DEFINITION  Vector for sgRNA expression in Acinetobacter baumannii (CRISPRi). 
            Modified version of pYDE007 (Plasmid #194152) made Golden Gate 
            compatible (BsaI) for sgRNA oligo insertion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8836)
  AUTHORS   Hamami E, Huo W, Neal K, Neisewander I, Geisinger E, Isberg RR
  TITLE     Dysfunction from loss of essential outer membrane functions causes 
            drug hypersensitization in Acinetobacter baumannii overexpressing 
            multidrug efflux pumps.
  JOURNAL   bioRxiv [Preprint]. 2025 Aug 4:2024.01.04.574119. doi: 
            10.1101/2024.01.04.574119.
  PUBMED    38260615
REFERENCE   2  (bases 1 to 8836)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 21, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Aug 4:2024.01.04.574119. doi: 
            10.1101/2024.01.04.574119."
COMMENT     Sequence Label: pEHE51
FEATURES             Location/Qualifiers
     source          1..8836
                     /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(join(854..1645,1646..1714))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1477..1496
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1715..1819)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1887..1905
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1932..1937,1938..1954,1955..1960)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        2308..2342
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        2363..2438
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2459..2488
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(8497..8519)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    8605..8745
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     8660..8679
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(8760..8777)
                     /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 cgcgagatcc acgctcaccg gctccagatt
     1021 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1081 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1141 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     1201 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1261 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1321 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1381 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1441 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     1501 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     1561 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     1621 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     1681 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     1741 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     1801 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     1861 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
     1921 aattctcatg tttgacagct tatcatcgat aagctttaat gcggtagttt atcacagtta
     1981 aattgctaac gcagtcaggc accgtgtatg aaatctaaca atgcgctcat cgtcatcctc
     2041 ggcaccgtca ccctggatgc tgtaggcata ggcttggtta tgccggtact gccgggcctc
     2101 ttgcgggata tcgtccattc cgacagcatc gccagtcact atggcgtgct gctagcgcta
     2161 tatgcgttga tgcaatttct atgcgcaccc gttctcggag cactgtccga ccgctttggc
     2221 cgccgcccag tcctgctcgc ttcgctactt ggagccacta tcgactacgc gatcatggcg
     2281 accacacccg tcctgtggat ccgatctttg acagctagct cagtcctagg tataatacta
     2341 gttgagaccg gatacggtct cagttttaga gctagaaata gcaagttaaa ataaggctag
     2401 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaagc ttgggcccga
     2461 acaaaaactc atctcagaag aggatctgaa tagcgccgtc gaccgatgcc cttgagagcc
     2521 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     2581 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     2641 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     2701 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     2761 aagcaggcca ttatcgccgg catggcggcc gacgcgctgg gctacgtctt gctggcgttc
     2821 gcgacgcgag gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg
     2881 atgcccgcgt tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt
     2941 caaggatcgc tcgcggctct taccagccta acttcgatca ttggaccgct gatcgtcacg
     3001 gcgatttatg ccgcctcggc gagcacatgg aacgggttgg catggattgt aggcgccgcc
     3061 ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc
     3121 tgaatggaag ccggcggcac ctcgctaacg gattcaccac tccaagaatt ggagccaatc
     3181 aattcttgcg gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt
     3241 ccgccatctc cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg
     3301 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     3361 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     3421 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3481 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3541 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     3601 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     3661 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     3721 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     3781 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     3841 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     3901 agctttaccg cagctgctgc tgttgctgct gttcaagtgt caattgctct actctgttgc
     3961 tcgtctcaat gatagacata tcaagccttt cagctttttt attgcgcttt ttgatttcat
     4021 cattacgctt gacgatctcc agttcctccc ccctgcgctt ggcgtgggtg gcttcaagtc
     4081 cctccttgaa tgtcggctcg atgtctagtc cacgatcctt atgactgcgg tgatcaactc
     4141 tcacctcaag ccctgctcgc tctaggtgga cgttcgtcag atccgccact ttttccctga
     4201 tttttttcag cgttgagttc tgatctagct ccctgacttt cttccctagc ccttgcggtg
     4261 taaggcttct tgttgtcatt agtatgtggg cgtgatggtt tctttcatca cttcccccat
     4321 gcacatgagg ggcatggatc gctacgtcca ccgccactcc ataggcttta actaaggact
     4381 ggcacaactc ggtaacaagt gcctgacgct gtgtcttatc cagttcatgc ggcaatgcta
     4441 tctctacttc ctttgctaat cttgcctcct gtttgatgtc accgttcttt tttagttcgg
     4501 attgctctac ccgattccat agggtttgac gatctaacat gtcaggactt gccccagttg
     4561 gggcaaaaat ttgggtgtat tcaatgcctg tttttttggt gtagtcctgc tcttttccgt
     4621 acgtatcaca gtacaatttt tcgcctgctc ggtatgctgc acatgccacg attgagcgac
     4681 catctgacct cgaaatgttc tgcatttcac aatggtaaat tgccattttc atcacctgtt
     4741 tattaacagc cgccccaagt tttgggcggt tggggtgtcg gggtttccct gaccgtactt
     4801 gcaaaatttg gcgtagcaaa aattttgcgt aagtgcgccc ttcgggaact ctgcgaggct
     4861 aaaagcaaaa gcaagagcaa taaggctttg actttgtttt ttgattttgc tcgctgcgct
     4921 cggatcttga tgtaagtcga gtttttgaag tagaacactt ttcacattga tgatggttca
     4981 tacttcggaa aataggttga gacacaggca ttaaaaatgg tcaaaaaaac agctatggaa
     5041 ttgggtcaaa tgctcgatga agaaaaggaa aaattggaac gtcaggaaaa ggcaagaaag
     5101 gatctagctg acgctgtggt gaaaggtcga gagcaaaaac aggcaaggtc agacgatgca
     5161 aaacgcaaga tcctgatcgg ggcttactta ctgaagaaac atgataatga catcaagaaa
     5221 cttgtggaac aaaaccccga ttttattggg tacattagag aaaatgataa gcacttattt
     5281 tcataggcat tagtagttat aaaaaaccct cataaatcga gggtttcttt tttatctgca
     5341 ttactttttg aatcaatgta cttttttagc ttgctatttt tcactgcata cagccgctta
     5401 tactctttct caatcaatgc ggtcaaaatg gctttttggc tctcccctgc tgcgcccacc
     5461 atgtccgcaa gcatatccga cacaccctta tctataaaca cctccaaacg ttgactatca
     5521 aggcttttgc gtttttcacg atatgctttt tgtcgttcgg cattggtttt tggcggctct
     5581 tttagcatgt cctgtgtttt ctgatcgttc gggtctttca tgtctttaac tcccatatag
     5641 tatatcatag ttacaggata acattgtttt tagttacagg ataacttttt ataaaattta
     5701 tttgtaaaaa tttatgctat cctgcaacta tttttaaatt acaggataac tatttaacgt
     5761 taccctgtaa ctatagttat cctgtaacgc taaattaatt accctgtaac atatattttg
     5821 ttatcctgta acaactaaat attgaaataa tactaccaaa gttaccctgt aacgaaaata
     5881 actaggttaa aaaatgatcg gattttaaca ttttgcgttg ttccaaaagt tatcaacagc
     5941 ctagaacgtc ataggaagcg attacagaca ctttatagct atcagcatgg gaacataagg
     6001 gcaggatgaa atatgggtct taaaacgcaa atggtgaggt tttagaggta ttttttgaag
     6061 atgattaagg cggtttgttt ttaaaatttt tggcggctct caggctgctt acattttaac
     6121 cagttcagtg aaaagttctt tttcagcaaa tttctgttta gcaccatagc taaaacttgc
     6181 gtggaacata ttaagaattg accgaaatga cacaatctca attatatttt ttttgaaaag
     6241 ttttctttat caaatatttt aaatcattga tttatatata agtatacatt cattttaata
     6301 attaatcttt atttaacaat gatttatcta tattcaattg tttaattatt cttactaata
     6361 ttatctctat atcaatattt tttatttaaa aacatatgtt tagtagtgct tttgattaaa
     6421 gtaccagagg gagggagcag agctgaatgg gaaatactca ccctagagcg attcttaaaa
     6481 atcaccctaa agtattccca ttcgatgtac cgtcggtcgg tcgctttcgc atcagggatg
     6541 acatcactgt atcaagctgc cactgttatg attacgattg atagcaccgc ctgaacacgc
     6601 tcataaccgc caattaaatg actactcatt gcgtccgcta ctctgttcag ttcccctagt
     6661 aatagcgttt ttccgatgtg tgctagcgtc actgtacctc atcacccaca catggacaga
     6721 ttgagttaca gacattggct aaatttttgg ggtctatgct tgacaaagcg agttaaaacc
     6781 ttagaattta agacaggtac attaagcccc tgtggtgaaa tcattagcgg tcgtcaaacc
     6841 ttaatgcttt cgattgccga tatgtcagta tcgaagatct gtaccccata aattacggta
     6901 aagccccaag caattgcaag gggcttttat cttttttaac aaaaaaaatt tataaatcag
     6961 gattttataa cactaaataa tccaaagtac atgagtaagt catgaccact cctgcgatgt
     7021 gtgtagcact ctgagtatcc gtattatatc agtgatgtca tatacaacca cataatgcgg
     7081 atgtatcact aactccctag tatctttctg tctgcctgtg cgccccatca gtcgattatc
     7141 aacaagtaaa tctgtctttt cttcaattaa atcatcaatt tcaatagctg ctatagggtt
     7201 gcgttcttca agataatcat agatatttct acgatcttgg cgagctgttt cagtccactc
     7261 aatcttcatt cttcgctgcc agtttcgcca atgttgcagc acgtctagcg gcaaactctg
     7321 cctttacgtc ctcatgtgga atcaacttgc ctgcattggc ttcatttaag ccgatctgta
     7381 cctgctgctt gaaccatttg tcatagtctg cggcttcctg ttgcttctta acaaagtcac
     7441 gcatgaagtc acggattagc tgcgcccccg accgatcaac agtcttagca aggcttgaaa
     7501 attcttgctt taggtcatgg tctacccgaa aggtaaaagt cgcttctgtc atgtttttca
     7561 ctccaatgtg ttacatttga atgctattgt aacacattaa aaagttaggt cattgtcttt
     7621 tttcggctta ggctgttctt ggtcataacg tggtttaaat gatggctgtc gctcattctc
     7681 aagtaaaatt ttccgtttat taacagcctg ttgcatctgc tcatagtaag gcatcaactg
     7741 cggtaaaata tcagcaagtg cctgtctgtc tttatccagg atacggctca attgcccgat
     7801 ttttttcaag tcacgctcat aggctattct gtcgctgatc tgctcgtatt tttgctttga
     7861 acaccttatg tactcttgca tatcgttact tgcccatgcc tgcgcctgaa gacgtgtaaa
     7921 catggaaatg tctgaagcaa catggtcgct gatatggtca tactcagcat ctttgacata
     7981 gccgtatttt gataaatatc tttcatagtc ctgccgtttc tttttgtcat cttcctgttt
     8041 ttgtttagtt tctctttcta tctgctgttg ttttcgttgc tgttcctgtt gctcctcttt
     8101 tatctctctg tgataaatgg aaaggctaga ttgatcatta tcccaacgtg tgagcatgtc
     8161 ctcaaagccg taataatcag cactaaacca acctgttttc actttcactg gtggcggcag
     8221 tggctctccg atctccctta gcagtgctgt agctgctcgt ccctctttga tcactttatc
     8281 aagctgcaag cggtcgatat tgtcttgcaa agccttgtaa aatctatgct ctgctcgctt
     8341 ctcatccgtt tttacttcaa tcaaatacac atctataagc gtttggcggt gttgttggtg
     8401 gtcgtatagt gcctgttgtg cggtggcata gactgcgggg gcttggtcta cggcatcctc
     8461 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     8521 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     8581 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     8641 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     8701 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     8761 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     8821 aaggcggtaa tacggt
//
