LOCUS       sequence-337259-        4709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PICASSO: Expression scaffold for second subcloning step to properly 
            co-express paired dCas9-fusion+sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4709)
  AUTHORS   Barber KW, Shrock E, Elledge SJ
  TITLE     CRISPR-based peptide library display and programmable microarray 
            self-assembly for rapid quantitative protein binding assays.
  JOURNAL   Mol Cell. 2021 Aug 4. pii: S1097-2765(21)00596-7. doi: 
            10.1016/j.molcel.2021.07.027.
  PUBMED    34390675
REFERENCE   2  (bases 1 to 4709)
  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: "Mol Cell. 
            2021 Aug 4. pii: S1097-2765(21)00596-7. doi: 
            10.1016/j.molcel.2021.07.027."
COMMENT     Sequence Label: Expression scaffold for dCas9+sgRNA
FEATURES             Location/Qualifiers
     source          1..4709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(112..139)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(155..226)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(247..264)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(271..297)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(391..409)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(392..409)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(427..443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(427..443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(440..462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    451..467
                     /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(475..481,482..499,500..505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(658..675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(829..1417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(909..928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1555..1929)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      1926..1935
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2135..2929)
                     /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             complement(2135..2929)
                     /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(2246..2265)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2856..2875
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3278..3580)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3380..3399)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3703..3725
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3717..3734
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3718..3734
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3741..3760
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3741..3759
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3838..3872)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     promoter        3912..4014
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4015..4674
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4081..4100)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      join(4695..4709,1..80)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
       61 cgttttttat tggtgagaat ccaagcagcg gaatatatcc ctaggtataa acgcagaaag
      121 gcccacccga aggtgagcca gtgtgactct agtagagagc gttcaccgac aaacaacaga
      181 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggagatcc
      241 ttattaatga tggtgatgat ggtgaccacc cgcgtagtcc ggaacgtcgt acgggtagtc
      301 gacgactgac taagggcgaa ttccagcaca ctggcggccg ttactagtgg atccgagctc
      361 ggtaccaagc ttgatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta
      421 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
      481 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
      541 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
      601 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
      661 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
      721 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
      781 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
      841 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
      901 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
      961 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1021 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1081 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1141 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1201 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1261 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1321 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1381 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1441 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1501 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttagca cgtgtcagtc
     1561 ctgctcctcg gccacgaagt gcacgcagtt gccggccggg tcgcgcaggg cgaactcccg
     1621 cccccacggc tgctcgccga tctcggtcat ggccggcccg gaggcgtccc ggaagttcgt
     1681 ggacacgacc tccgaccact cggcgtacag ctcgtccagg ccgcgcaccc acacccaggc
     1741 cagggtgttg tccggcacca cctggtcctg gaccgcgctg atgaacaggg tcacgtcgtc
     1801 ccggaccaca ccggcgaagt cgtcctccac gaagtcccgg gagaacccga gccggtcggt
     1861 ccagaactcg accgctccgg cgacgtcgcg cgcggtgagc accggaacgg cactggtcaa
     1921 cttggccatg gtggccctcc tcacgtgcta ttattgaagc atttatcagg gttattgtct
     1981 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2041 atttccccga aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     2101 aataccgcat caggaaattg taagcgttaa taattcagaa gaactcgtca agaaggcgat
     2161 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     2221 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     2281 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     2341 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     2401 tcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     2461 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     2521 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     2581 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     2641 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     2701 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc agttcattca
     2761 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     2821 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     2881 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     2941 ctcatcctgt ctcttgatca gagcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     3001 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     3061 attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac
     3121 tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc
     3181 tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg aaaaggatct
     3241 aggtgaagat cctttttgat aatctcatgc ctgacattta tattccccag aacatcaggt
     3301 taatggcgtt tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa
     3361 cggagaccgg cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat
     3421 gcaccaccgg gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg
     3481 ggatcaccat ccgtcgcccc ggcgtgtcaa taatatcact ctgtacatcc acaaacagac
     3541 gataacggct ctctctttta taggtgtaaa ccttaaactg ccgtacgtat aggctgcgca
     3601 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     3661 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     3721 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggc cctctagatg
     3781 catgctcgag cggccgccag tgtgatggat atctgcagaa ttcgccctta tgcatgcact
     3841 agtattatac ctaggactga gctagctgtc aaagatcttt aggacgtcga tatctggcga
     3901 aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca
     3961 ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag
     4021 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     4081 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     4141 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     4201 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     4261 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     4321 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     4381 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     4441 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     4501 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     4561 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     4621 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttgat
     4681 atcgagctcg cttggactcc tgttgatag
//
