LOCUS       sequence-182682-        5284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Receptor plasmid for the assembly of multiple sgRNAs with DsRed 
            coexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5284)
  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 5284)
  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. 2017 Sep 7. doi: 10.1021/acssynbio.7b00268."
COMMENT     Sequence Label: pMulti-sgRNA-LacZ-DsRed
FEATURES             Location/Qualifiers
     source          1..5284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..46)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     81..100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    267..288
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(303..308,309..326,327..333)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    341..357
                     /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     346..368
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     365..381
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     365..381
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             377..793
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLEDNRKLVNCKR"
     primer_bind     400..420
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        402..420
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(467..486)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(468..486)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(496..513)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(496..512)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(505..527)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(778..1233)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(915..936)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1127..1146
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1260..1364
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1366..1723
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1366..1557
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1389..1409)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1574..1709
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1758..2552
                     /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             1758..2552
                     /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(1812..1831)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2422..2441
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2740..2759)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2784..2831
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3160..3748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3649..3668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3798..4101
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4102..4305
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4256..4276
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4470..4479
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4476..5153
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Strongin et al., 2007)"
                     /label=DsRed-Monomer
                     /note="mammalian codon-optimized"
                     /translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV
                     VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH
                     ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS
                     GSQ"
     polyA_signal    join(5274..5284,1..111)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
       61 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtctg
      121 ccttgtcttc cttctgatca attccggcgc gtgcggcaat ccctttttca ctgcatccat
      181 ggcaggacgt ccgccgaggc aggccaaggc gacgaggtgg tcgttggaga cgaccggtgg
      241 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      301 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      361 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      421 gaacaaaagc tggaggacct cgaggggggg cccggtaccc aattcgccct atagtgagtc
      481 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
      541 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
      601 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
      661 ctgtagcggc gcccgtctcc aggggggcac ccgtcagtgc attgcgcgat gcatgcactg
      721 cctccattgc ggtcacgccg ccacgttttg caatcttgaa gacaacagaa agctcgtaaa
      781 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
      841 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
      901 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
      961 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     1021 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     1081 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     1141 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     1201 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     1261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     1381 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     1441 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     1501 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     1561 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     1621 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     1681 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     1741 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     1801 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     1861 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     1921 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     1981 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     2041 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     2101 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     2161 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     2221 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     2281 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     2341 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     2401 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     2461 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     2521 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     2581 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     2641 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     2701 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     2761 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     2821 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     2881 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     2941 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     3001 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3061 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3121 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3181 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3241 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3301 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3361 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3421 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3481 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3541 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3601 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3661 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3721 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt ggttcatagc
     3781 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     3841 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     3901 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     3961 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4021 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     4081 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     4141 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     4201 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     4261 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     4321 cagatccgct agcgctaccg gactcagatc tcgaccgagt tccccgcgcc agcggggtta
     4381 aaccgcgccc gccagcttcg aattctgcag tcgacggtac cgcgggccga gttccccgcg
     4441 ccagcggggt taaaccggat ccaccggtcg ccaccatgga caacaccgag gacgtcatca
     4501 aggagttcat gcagttcaag gtgcgcatgg agggctccgt gaacggccac tacttcgaga
     4561 tcgagggcga gggcgagggc aagccctacg agggcaccca gaccgccaag ctgcaggtga
     4621 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct
     4681 ccaaggccta cgtgaagcac cccgccgaca tccccgacta catgaagctg tccttccccg
     4741 agggcttcac ctgggagcgc tccatgaact tcgaggacgg cggcgtggtg gaggtgcagc
     4801 aggactcctc cctgcaggac ggcaccttca tctacaaggt gaagttcaag ggcgtgaact
     4861 tccccgccga cggccccgta atgcagaaga aaactgccgg ctgggagccc tccaccgaga
     4921 agctgtaccc ccaggacggc gtgctgaagg gcgagatctc ccacgccctg aagctgaagg
     4981 acggcggcca ctacacctgc gacttcaaga ccgtgtacaa ggccaagaag cccgtgcagc
     5041 tgcccggcaa ccactacgtg gactccaagc tggacatcac caaccacaac gaggactaca
     5101 ccgtggtgga gcagtacgag cacgccgagg cccgccactc cggctcccag tagcggccgc
     5161 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     5221 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     5281 ttat
//
