LOCUS       sequence-403768-        3672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  universal template to build tRNA-gRNA array for multiplex editing 
            CREEPY.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3672)
  AUTHORS   Zhao Y, Coelho C, Lauer S, Majewski M, Laurent JM, Brosh R, Boeke JD
  TITLE     CREEPY: CRISPR-mediated editing of synthetic episomes in yeast.
  JOURNAL   Nucleic Acids Res. 2023 Jun 16:gkad491. doi: 10.1093/nar/gkad491.
  PUBMED    37326023
REFERENCE   2  (bases 1 to 3672)
  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: "Nucleic 
            Acids Res. 2023 Jun 16:gkad491. doi: 10.1093/nar/gkad491."
COMMENT     Sequence Label: pYZ038
FEATURES             Location/Qualifiers
     source          1..3672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(908..1282)
                     /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      1279..1288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1488..2282)
                     /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(1488..2282)
                     /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(1599..1618)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2209..2228
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2631..2933)
                     /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(2733..2752)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3056..3078
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3070..3087
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3071..3087
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3094..3113
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3094..3112
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        3184..3259
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3416..3434)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3417..3434)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3452..3468)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3452..3468)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3465..3487)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3476..3492
                     /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(3500..3506,3507..3524,3525..3530))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3545..3566
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 gcacgtgtca gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca
      961 gggcgaactc ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt
     1021 cccggaagtt cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca
     1081 cccacaccca ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca
     1141 gggtcacgtc gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc
     1201 cgagccggtc ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa
     1261 cggcactggt caacttggcc atggtggccc tcctcacgtg ctattattga agcatttatc
     1321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1381 gggttccgcg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga
     1441 tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taataattca gaagaactcg
     1501 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     1561 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     1621 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     1681 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     1741 ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     1801 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     1861 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     1921 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     1981 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     2041 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct
     2101 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     2161 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     2221 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     2281 atgcgaaacg atcctcatcc tgtctcttga tcagagcttg atcccctgcg ccatcagatc
     2341 cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
     2401 gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc
     2461 catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag
     2521 atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac
     2581 gtgaaaagga tctaggtgaa gatccttttt gataatctca tgcctgacat ttatattccc
     2641 cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact
     2701 tcttccccga taacggagac cggcacactg gccatatcgg tggtcatcat gcgccagctt
     2761 tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt
     2821 gcactggcca gggggatcac catccgtcgc cccggcgtgt caataatatc actctgtaca
     2881 tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgccgtacg
     2941 tataggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     3001 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     3061 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg
     3121 ggccctctag atgcatgctc gagcggccgc cagtgtgatg gatatctgca gaattcgccc
     3181 ttagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     3241 agtggcaccg agtcggtgca aacaagcgca agtggtttag tggtaaaatc caacgttgcc
     3301 atcgttgggc ccccggttcg attccgggct tgcgcaaagg gcgaattcca gcacactggc
     3361 ggccgttact agtggatccg agctcggtac caagcttgat gcatagcttg agtattctat
     3421 agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3481 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3541 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3601 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3661 gtattgggcg ct
//
