LOCUS       sequence-384916-        2710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 MoClo plasmid for Golden Gate cloning of sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2710)
  AUTHORS   Brychcy M, Kokodynski A, Lloyd D, Godoy VG
  TITLE     AspFlex: Molecular Tools to Study Gene Expression and Regulation in 
            Acinetobacter baumannii.
  JOURNAL   ACS Synth Biol. 2023 Aug 16. doi: 10.1021/acssynbio.3c00167.
  PUBMED    37587063
REFERENCE   2  (bases 1 to 2710)
  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: "ACS Synth 
            Biol. 2023 Aug 16. doi: 10.1021/acssynbio.3c00167."
COMMENT     Sequence Label: pBP_sgRNA
FEATURES             Location/Qualifiers
     source          1..2710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2..59
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(83..100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(254..842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(334..353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1025..1119)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1140..1799)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1714..1733
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     1961..1979
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1983..2026)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2029..2048
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     promoter        2072..2106
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             complement(2149..2457)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMLEPSSARHWDIPIRVNSLAVVLQRRDWENPGVTQLNRLAAHP
                     PFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     primer_bind     2154..2173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2367..2389
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2381..2398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2382..2398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2453..2469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2453..2469)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2466..2488)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2477..2493
                     /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(2501..2507,2508..2525,2526..2531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2546..2567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        2592..2667
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gtccggcaaa aaagggcaag gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat
       61 tacttcgcgt tatgcaggct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      241 cgttgctggc gtttttccac aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agctcgaggc
     1021 ttggattctc accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat
     1081 ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc gatatcaaat
     1141 tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca
     1201 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
     1261 ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc
     1321 acgtttaaat caaaactggt gaaactcacc cagggattgg ctgacacgaa aaacatattc
     1381 tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa
     1441 tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt
     1501 tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca
     1561 ccgtctttca ttgccatacg aaattccgga tgagcattca tcaggcgggc aagaatgtga
     1621 ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata
     1681 tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
     1741 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
     1801 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
     1861 atttcattat ggtgaaagtt ggaacctctt acgtgcccga tcaactcgag tgccacctga
     1921 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggca
     1981 gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgga attcgcggcc
     2041 gcttctagaa tcttggtctc tctatcgatc tttgacagct agctcagtcc taggtataat
     2101 actagtaagt cttccatatg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2161 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     2221 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     2281 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     2341 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     2401 attgacgcgt attgggatat cccaatggcg cgccgagctt ggctcgagca tggtcatagc
     2461 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2521 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaacg cactagtgga
     2581 tccgaagaca agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     2641 cttgaaaaag tggcaccgag tcggtgcttt ttttgaagct ttgtttgaga ccgcatgcaa
     2701 tttagctgca
//
