LOCUS       sequence-325466-        5269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmids containing the medium-copy-number p15A origin of 
            replication can be propagated in E. coli cells and a non-targeting 
            crRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 113 to 147)
  AUTHORS   Yan MY, Yan HQ, Ren GX, Zhao JP, Guo XP, Sun YC
  TITLE     CRISPR-Cas12a-Assisted Recombineering in Bacteria.
  JOURNAL   Appl Environ Microbiol. 2017 Aug 17;83(17). pii: AEM.00947-17. doi: 
            10.1128/AEM.00947-17. Print 2017 Sep 1.
  PUBMED    28646112
REFERENCE   2  (bases 1 to 5269)
  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: "Appl 
            Environ Microbiol."; date: "2017-08-17"; volume: "83(17). pii: 
            AEM.00947-17. doi"; pages: " 10.1128/AEM.00947-17. Print 2017 Sep 1"
COMMENT     Sequence Label: pAC-crRNA-Cm
FEATURES             Location/Qualifiers
     source          1..5269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..29)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        113..147
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     promoter        1018..1463
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1464..1550,1551..2885)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFVKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             complement(3325..3984)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3899..3918
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3985..4087)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4479..4496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4613..5158)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4693..4712)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgacagctt atcatcgata agctttaatg cggtagttta tcacagttaa attgctagtc
       61 taagaacttt aaataatttc tactgttgta gatgagacct ttcctgctcc agttgacggc
      121 tagctcagtc ctaggtacag tgctagctac tagagtcaca caggaaagta ctagatgtca
      181 gtgattaagc aggtaatgaa gaccaagttg caccttgagg gcactgtcaa tggccatgat
      241 tttacgatcg agggtaaagg tgaaggcaag ccgtacgaag ggttacagca catgaaaatg
      301 acagtcacca aaggcgcgcc tctgccgttt tccgttcata ttcttacacc tagccacatg
      361 tatggaagca aaccgtttaa taagtatcca gcggatatcc cagactacca caaacagtct
      421 tttcccgaag gtatgtcttg ggagcggtcg atgatttttg aagatggtgg cgtatgcacc
      481 gccagtaatc attccagcat aaacttgcaa gagaactgtt tcatctatga tgttaaattt
      541 catggtgtga acctgcctcc ggatgggccc gtaatgcaaa aaaccattgc tggatgggag
      601 ccgagcgtgg aaacactgta cgtgcgtgac gggatgttaa aaagtgacac tgcaatggtt
      661 tttaaactga aaggaggcgg tcatcatcgt gttgatttca aaacgacgta taaagccaaa
      721 aaacctgtca agctgccaga atttcatttc gttgaacatc gcctggaact gaccaaacac
      781 gataaagatt tcacaacttg ggaccagcag gaggcagccg aaggccattt ctcaccgctg
      841 ccgaaggctc tcccataact ggagtgtgaa aggtctctgt ctaagaactt taaataattt
      901 ctactgttgt agatgtcagt ttcacctgtt ttacgtaaaa acccgcttcg gcgggttttt
      961 acttttggct cgagcattca aatatgtatc cgctcaccgc gatccttttt aacccatcac
     1021 atatacctgc cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt
     1081 gattaaaaag gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa
     1141 agaaacagat agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt
     1201 ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga
     1261 ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt
     1321 aaagggcaaa gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt
     1381 gcgtaactaa cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt
     1441 acataaaaaa ggagacatga acgatgaaca tcaaaaagtt tgtaaaacaa gcaacagtat
     1501 taacctttac taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga
     1561 accaaaagcc atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc
     1621 aaatccctga acagcaaaaa aatgaaaaat atcaagttcc tgagttcgat tcgtccacaa
     1681 ttaaaaatat ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaacg
     1741 ctgacggcac tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc
     1801 ctaaaaatgc ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta
     1861 ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg
     1921 attctatcct aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg
     1981 gaaaaatccg tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga
     2041 caactgcaca agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg
     2101 attataaatc aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg
     2161 atgaaggcaa ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag
     2221 ataaaggcca caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag
     2281 gcgaagaatc tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag
     2341 aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc
     2401 tcggtatgat tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg
     2461 catctaacac agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat
     2521 ggtacctgtt cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg
     2581 atatttacat gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca
     2641 aaactggcct tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac
     2701 acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca
     2761 gaggattcta cgcagacaaa caatcaacgt ttgcgccaag cttcctgctg aacatcaaag
     2821 gcaagaaaac atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca
     2881 aataaaaacg caaaagaaaa tgccgatatt gactaccgga agcagtgtga ccgtgtgctt
     2941 ctcaaatgcc tgattcaggc tgtctatgtg tgactgttga gctgtaacaa gttgtctcag
     3001 gtgttcaatt tcatgttcta gttgctttgt tttactggtt tcacctgttc tattaggtgt
     3061 tacatgctgt tcatctgtta cattgtcgat ctgttcatgg tgaacagctt taaatgcacc
     3121 aaaaactcgt aaaagctctg atgtatctat cttttttaca ccgttttcat ctgtgcatat
     3181 gataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga
     3241 atttctgcca ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg
     3301 gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg
     3361 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     3421 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     3481 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     3541 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     3601 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     3661 ttcactccaa agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     3721 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
     3781 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     3841 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     3901 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     3961 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     4021 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     4081 ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac
     4141 accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag
     4201 tgcgtcgggt gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctt
     4261 cagtggcttc tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta
     4321 acggcaaaag caccgccgga catcagcgct agcggagtgt atactggctt actatgttgg
     4381 cactgatgag ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg
     4441 cgtcagcaga atatgtgata caggatatat tccgcttcct cgctcactga ctcgctacgc
     4501 tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa
     4561 gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata
     4621 ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     4681 cgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg
     4741 ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca
     4801 cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc
     4861 cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa
     4921 agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt
     4981 gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc
     5041 aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg
     5101 caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga
     5161 agatcatctt attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa
     5221 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgt
//
