LOCUS       sequence-225141-        6649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For PYL expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6649)
  AUTHORS   Yang B, Borgeaud AC, Buricova M, Aeschbach L, Rodriguez-Lima O, Ruiz
            Buendia GA, Cinesi C, Taylor AS, Baubec T, Dion V
  TITLE     Expanded CAG/CTG repeats resist gene silencing mediated by targeted 
            Epigenome editing.
  JOURNAL   Hum Mol Genet. 2021 Sep 7. pii: 6365982. doi: 10.1093/hmg/ddab255.
  PUBMED    34494094
REFERENCE   2  (bases 1 to 6649)
  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: "Hum Mol 
            Genet. 2021 Sep 7. pii: 6365982. doi: 10.1093/hmg/ddab255."
COMMENT     Sequence Label: pAB-NEO-PYL
FEATURES             Location/Qualifiers
     source          1..6649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        480..683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     634..654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     728..747
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        728..746
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      778..787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1321..1392
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             1396..1416
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    1417..1541
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1574..1876
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1758..1777
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2018..2042)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2067..2085)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2068..2085)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2105..2122)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2111..2335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2381..2809
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2468..2487)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2678..2699
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2818..2838)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2823..3152
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3003..3138
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3065..3084
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3219..4013
                     /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(3273..3292)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3883..3902
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4187..4308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4224..4243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4278..4297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4357..4373)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4357..4373)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4370..4392)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4381..4397
                     /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(4405..4411,4412..4429,4430..4435))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4450..4471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4588..4605)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4759..5347)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4839..4858)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5518..6309,6310..6378))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6141..6160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6379..6483)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6558..6577)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
       61 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      661 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
      721 tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgagct cggatccgcc
      781 accatggcca cccaggacga gttcacccag ctgagccagt ctatcgccga gttccacacc
      841 taccagctgg gcaacggccg gtgcagcagc ctgctggccc agagaatcca cgcccctccc
      901 gagacagtgt ggtccgtcgt gcggagattc gaccggcccc agatctacaa gcacttcatc
      961 aagagctgca acgtgtccga ggacttcgag atgagagtgg gctgcacccg ggacgtgaac
     1021 gtgatcagcg gcctgcccgc caacaccagc agagagagac tggacctgct ggacgacgac
     1081 agaagagtga ccggcttcag catcaccggc ggagagcacc ggctgcggaa ctacaagagc
     1141 gtgaccaccg tgcacagatt cgagaaagag gaagaagagg aacggatctg gaccgtggtg
     1201 ctggaaagct acgtggtgga cgtgcccgag ggcaacagcg aagaggacac ccggctgttc
     1261 gccgacaccg tgatccggct gaacctgcag aagctggcct ccatcaccga ggccatgaac
     1321 gactacaagg acgacgacga caaggactat aaggacgatg atgataagga ttacaaagat
     1381 gacgacgata agccccccaa gaagaagcgg aaagtcacaa gtttgtacaa aaaagctgaa
     1441 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     1501 actacataat actgtaaaac acaacatatc cagtcactat gcaccaggct ttacacttta
     1561 tgcttccggc tcgatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
     1621 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
     1681 ggccagtgca cgtctgctgt cagataaagt cccccgtgaa ctttacccgg tggtgcatat
     1741 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat
     1801 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
     1861 gatgttctgg ggaataatgt caggctccct tatacacagc cagtctgcag gtcgaccata
     1921 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     1981 ttaatatatt gatatttata tcattttacg tttctcggtt cagctttctt gtacaaagtg
     2041 gtcatccgcg gtctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga
     2101 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2161 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2221 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2281 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2341 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2401 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2461 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2521 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2581 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2641 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2701 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2761 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2821 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2881 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2941 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3001 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3061 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3121 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3181 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3241 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3301 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3361 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3421 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3481 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3541 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3601 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3661 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3721 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3781 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3841 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3901 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3961 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4021 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4081 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4141 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4201 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4261 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4321 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4381 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4441 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4501 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4561 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4621 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4681 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4741 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4801 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4861 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4921 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4981 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5041 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5101 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5161 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5221 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5281 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5341 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5401 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5461 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5521 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5581 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5641 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5701 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5761 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5821 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5881 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5941 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6001 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6061 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6121 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6181 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6241 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6301 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6361 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6421 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6481 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6541 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6601 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaag
//
