LOCUS       sequence-431928-        6110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses crRNA for genome editing in Acinetobacter baumannii.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6110)
  AUTHORS   Wang S, Ding Y, Rong H, Wang Y
  TITLE     The Development of a CRISPR-FnCpf1 System for Large-Fragment 
            Deletion and Multiplex Gene Editing in Acinetobacter baumannii.
  JOURNAL   Curr Issues Mol Biol. 2024 Jan 5;46(1):570-584. doi: 
            10.3390/cimb46010037.
  PUBMED    38248339
REFERENCE   2  (bases 1 to 6110)
  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"; journalName: 
            "Curr Issues Mol Biol"; date: "2024-01-5- 5"; volume: "46"; issue: 
            "1"; pages: "570-584"
COMMENT     Sequence Label: pCrAb-km
FEATURES             Location/Qualifiers
     source          1..6110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1708..1730
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1722..1739
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1723..1739
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1776..1792
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1777..1793
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(1855..3189,3190..3276))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        3654..3688
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     ncRNA           3725..3792
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(3859..3875)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3859..3875)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3934..3950)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3934..3950)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3947..3969)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3958..3974
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3982..3988,3989..4006,4007..4012))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4027..4048
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4165..4182)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4336..4924)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4416..4435)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5095..5910)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5095..5910)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5818..5837
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5911..6015)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6083..6101
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataggat tttaacattt tgcgttgttc caaaagttat caacagccta gaacgtcata
      241 ggaagcgatt acagacactt tatagctatc agcatgggaa cataagggca ggatgaaata
      301 tgggtcttaa aacgcaaatg gtgaggtttt agaggtattt tttgaagatg attaaggcgg
      361 tttgttttta aaatttttgg cggctctcag gctgcttaca ttttaaccag ttcagtgaaa
      421 agttcttttt cagcaaattt ctgtttagca ccatagctaa aacttgcgtg gaacatatta
      481 agaattgacc gaaatgacac aatctcaatt atattttttt tgaaaagttt tctttatcaa
      541 atattttaaa tcattgattt atatataagt atgcattcat tttaataatt aatctttatt
      601 taacaatgat ttatctatat tcaattgttt aattattctt actaatatta tctctatatc
      661 aatatttttt atttaaaaac atatgtttag tagtgctttt gattaaagta ccagagggag
      721 ggagcagagc tgaatgggaa atactcaccc tagagcgatt cttaaaaatc accctaaagt
      781 attcccattc gatgtaccgt cggtcggtcg ctttcgcatc agggatgaca tcactgtatc
      841 aagctgccac tgttatgatt acgattgata gcaccgcctg aacacgctca taaccgccaa
      901 ttaaatgact actcattgcg tccgctactc tgttcagttc ccctagtaat agcgtttttc
      961 cgatgtgtgc tagcgtcact gtacctcatc acccacacat ggacagattg agttacagac
     1021 attggctaaa tttttggggt ctatgcttga caaagcgagt taaaacctta gaatttaaga
     1081 caggtacatt aagcccctgt ggtgaaatca ttagcggtcg tcaaacctta atgctttcga
     1141 ttgccgatat gtcagtatcg aagatctgta ccccataaat tacggtaaag ccccaagcaa
     1201 ttgcaagggg cttttatctt ttttaacaaa aaaaatttat aaatcaggat tttataacac
     1261 taaataatcc aaagtacatg agtaagtcat gaccactcct gcgatgtgtg tagcactctg
     1321 agtatccgta ttatatcagt gatgtcatat acaaccacat aatgcggatg tatcactaac
     1381 tccctagtat ctttctgtct gcctgtgcgc cccatcagtc gattatcaac aagtaaatct
     1441 gtcttttctt caattaaatc atcaatttca atagctgcta tagggttgcg ttcttcaaga
     1501 taatcataga tatttctacg atcttggcta tgcggtgtga aataccgcac agatgcgtaa
     1561 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     1621 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     1681 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     1741 aattgacgcg tattgggatg gtaccgggcc ccccctcgag gtcgacggta tcgatacggc
     1801 atcagagcag attgtactga gagtgcacca taatcggcat tttcttttgc gtttttattt
     1861 gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg ttttcttgcc
     1921 tttgatgttc agcaggaagc taggcgcaaa cgttgattgt ttgtctgcgt agaatcctct
     1981 gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta cagcgaagtg
     2041 tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa ggccagtttt
     2101 gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca tgtaaatatc
     2161 gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga acagatacca
     2221 tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg tgttagatgc
     2281 aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa tcataccgag
     2341 agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc
     2401 ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag attcttcgcc
     2461 ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt ggcctttatc
     2521 ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc
     2581 gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg atttataatc
     2641 ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt gtgcagttgt
     2701 cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac ggatttttcc
     2761 gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta ggatagaatc
     2821 atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat
     2881 agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg catttttagg
     2941 atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag tgccgtcagc
     3001 gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga tatttttaat
     3061 tgtggacgaa tcgaactcag gaacttgata tttttcattt ttttgctgtt cagggatttg
     3121 cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg gcttttggtt
     3181 cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag taaaggttaa
     3241 tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct tttttatgta
     3301 ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt tagttacgca
     3361 caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact ttgcccttta
     3421 cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac ttttcgacct
     3481 cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt gtttgataga
     3541 aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta tctgtttctt
     3601 ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgca agcttgacag
     3661 ctagctcagt cctaggtata atactagtaa tttctactgt tgtagatcga gaccattggt
     3721 ctcaggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat gcttcggcat
     3781 ggcgaatggg acccaattat tgaacaccct aacgggtgtt tttttgtttc tggtctaccg
     3841 aattcctgca gcccggggga tccactagtt ctagagcggc cgccaccgcg gtggagctca
     3901 tcccaatggc gcgccgagct tggctcgagc atggtcatag ctgtttcctg tgtgaaattg
     3961 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4021 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4081 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4141 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4201 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4261 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4321 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4381 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4441 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4501 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4561 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4621 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4681 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4741 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4801 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4861 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4921 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4981 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5041 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttagaa
     5101 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     5161 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     5221 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     5281 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     5341 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     5401 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     5461 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     5521 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     5581 taatacctgg aatgctgttt tcccagggat cgcagtggtg agtaaccatg catcatcagg
     5641 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     5701 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     5761 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     5821 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     5881 gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat
     5941 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6001 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     6061 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
