LOCUS       sequence-321925-        5440 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 5440)
  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-Km
FEATURES             Location/Qualifiers
     source          1..5440
                     /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]
     CDS             175..894
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     promoter        1054..1499
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1500..1586,1587..2921)
                     /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(3361..4155)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3472..3491)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4082..4101
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4156..4258)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4650..4667)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4784..5329)
                     /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(4864..4883)
                     /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 ctagatggtg
      181 agcaagggcg ccgagctgtt caccggcatc gtgcccatcc tgatcgagct gaatggcgat
      241 gtgaatggcc acaagttcag cgtgagcggc gagggcgagg gcgatgccac ctacggcaag
      301 ctgaccctga agttcatctg caccaccggc aagctgcctg tgccctggcc caccctggtg
      361 accaccctga gctacggcgt gcagtgcttc tcacgctacc ccgatcacat gaagcagcac
      421 gacttcttca agagcgccat gcctgagggc tacatccagg agcgcaccat cttcttcgag
      481 gatgacggca actacaagtc gcgcgccgag gtgaagttcg agggcgatac cctggtgaat
      541 cgcatcgagc tgaccggcac cgatttcaag gaggatggca acatcctggg caataagatg
      601 gagtacaact acaacgccca caatgtgtac atcatgaccg acaaggccaa gaatggcatc
      661 aaggtgaact tcaagatccg ccacaacatc gaggatggca gcgtgcagct ggccgaccac
      721 taccagcaga atacccccat cggcgatggc cctgtgctgc tgcccgataa ccactacctg
      781 tccacccaga gcgccctgtc caaggacccc aacgagaagc gcgatcacat gatctacttc
      841 ggcttcgtga ccgccgccgc catcacccac ggcatggatg agctgtacaa gtgactggag
      901 tgtgaaaggt ctctgtctaa gaactttaaa taatttctac tgttgtagat gtcagtttca
      961 cctgttttac gtaaaaaccc gcttcggcgg gtttttactt ttggctcgag cattcaaata
     1021 tgtatccgct caccgcgatc ctttttaacc catcacatat acctgccgtt cactattatt
     1081 tagtgaaatg agatattatg atattttctg aattgtgatt aaaaaggcaa ctttatgccc
     1141 atgcaacaga aactataaaa aatacagaga atgaaaagaa acagatagat tttttagttc
     1201 tttaggcccg tagtctgcaa atccttttat gattttctat caaacaaaag aggaaaatag
     1261 accagttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa tcgcgcgggt
     1321 ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt atactttggc
     1381 gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg ccatcttcaa
     1441 acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag acatgaacga
     1501 tgaacatcaa aaagtttgta aaacaagcaa cagtattaac ctttactacc gcactgctgg
     1561 caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat aaggaaacat
     1621 acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag caaaaaaatg
     1681 aaaaatatca agttcctgag ttcgattcgt ccacaattaa aaatatctct tctgcaaaag
     1741 gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc gcaaactatc
     1801 acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat gacacatcga
     1861 tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa aacgctggcc
     1921 gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa gaccaaacac
     1981 aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta ttctacactg
     2041 atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag
     2101 catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc tttgacggtg
     2161 acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac agctcaggcg
     2221 acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa tacttagtat
     2281 ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta tttaacaaag
     2341 catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg
     2401 ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg
     2461 attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta acagatgaaa
     2521 ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact gactcccgcg
     2581 gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt ggttatgttt
     2641 ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg
     2701 atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct caagcgaaag
     2761 gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca gacaaacaat
     2821 caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag
     2881 acagcatcct tgaacaagga caattaacag ttaacaaata aaaacgcaaa agaaaatgcc
     2941 gatattgact accggaagca gtgtgaccgt gtgcttctca aatgcctgat tcaggctgtc
     3001 tatgtgtgac tgttgagctg taacaagttg tctcaggtgt tcaatttcat gttctagttg
     3061 ctttgtttta ctggtttcac ctgttctatt aggtgttaca tgctgttcat ctgttacatt
     3121 gtcgatctgt tcatggtgaa cagctttaaa tgcaccaaaa actcgtaaaa gctctgatgt
     3181 atctatcttt tttacaccgt tttcatctgt gcatatgata agcggctatt taacgaccct
     3241 gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt
     3301 atcacttatt caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa
     3361 ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     3421 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     3481 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     3541 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     3601 gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
     3661 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     3721 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     3781 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     3841 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     3901 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     3961 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     4021 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     4081 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     4141 atcttgttca atcattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     4201 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     4261 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     4321 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg
     4381 tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct tcagtggctt
     4441 ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa
     4501 gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga
     4561 gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag
     4621 aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     4681 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg
     4741 aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc
     4801 cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac
     4861 tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct
     4921 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
     4981 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
     5041 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
     5101 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
     5161 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
     5221 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
     5281 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
     5341 tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc
     5401 tgaagtcagc cccatacgat ataagttgta attctcatgt
//
