LOCUS       sequence-194884-        3658 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Receptor plasmid for the assembly of multiple sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3658)
  AUTHORS   Shao S, Chang L, Sun Y, Hou Y, Fan X, Sun Y
  TITLE     Multiplexed sgRNA Expression Allows Versatile Single Nonrepetitive 
            DNA Labeling and Endogenous Gene Regulation.
  JOURNAL   ACS Synth Biol. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268.
  PUBMED    28849913
REFERENCE   2  (bases 1 to 3658)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268."
COMMENT     Sequence Label: pMulti-sgRNA-LacZ
FEATURES             Location/Qualifiers
     source          1..3658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    253..274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(289..294,295..312,313..319)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    327..343
                     /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)."
     primer_bind     332..354
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     351..367
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     351..367
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             363..779
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLEDNRKLVNCKR"
     primer_bind     386..406
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        388..406
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(453..472)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(454..472)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(482..499)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(482..498)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(491..513)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(764..1219)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(901..922)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1113..1132
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1246..1350
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1352..1709
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1352..1543
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1375..1395)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1560..1695
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1744..2538
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1744..2538
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1798..1817)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2408..2427
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2726..2745)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2770..2817
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      join(3146..3658,1..76)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3635..3654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
       61 gcggagccta tggaaaaacg ccagcaacgc ggccttttga attctgcctt gtcttccttc
      121 tgatcaattc cggcgcgtgc ggcaatccct ttttcactgc atccatggca ggacgtccgc
      181 cgaggcaggc caaggcgacg aggtggtcgt tggagacgac cggtggaaag cgggcagtga
      241 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      301 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      361 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
      421 ggacctcgag ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
      481 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
      541 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
      601 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgccc
      661 gtctccaggg gggcacccgt cagtgcattg cgcgatgcat gcactgcctc cattgcggtc
      721 acgccgccac gttttgcaat cttgaagaca acagaaagct cgtaaattgt aagcgttaat
      781 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
      841 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
      901 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
      961 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     1021 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     1081 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     1141 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     1201 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1261 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1321 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     1381 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     1441 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     1501 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     1561 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     1621 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     1681 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     1741 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     1801 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     1861 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     1921 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     1981 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     2041 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     2101 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     2161 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     2221 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     2281 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     2341 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     2401 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     2461 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     2521 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     2581 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     2641 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     2701 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     2761 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     2821 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     2881 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     2941 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     3001 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3061 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3121 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3181 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3241 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3301 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3361 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3421 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3481 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3541 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3601 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatag
//
