LOCUS       sequence-324991-        7705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For yeast genomic integration of sgRNA against HTS1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7705)
  AUTHORS   Muller R, Meacham ZA, Ferguson L, Ingolia NT
  TITLE     CiBER-seq dissects genetic networks by quantitative CRISPRi 
            profiling of expression phenotypes.
  JOURNAL   Science. 2020 Dec 11;370(6522). pii: 370/6522/eabb9662. doi: 
            10.1126/science.abb9662.
  PUBMED    33303588
REFERENCE   2  (bases 1 to 7705)
  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: "Science."; 
            date: "2020-12-11"; volume: "370(6522). pii: 370/6522/eabb9662. 
            doi"; pages: " 10.1126/science.abb9662"
COMMENT     Sequence Label: pNTI732 pRPR1(TetO)-HTS1-sgRNA
FEATURES             Location/Qualifiers
     source          1..7705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      19..208
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    596..614
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        699..774
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      787..849
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     protein_bind    966..999
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3288..3321
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3364..3383)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3365..3383)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3393..3409)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3402..3424)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(4072..4105)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(4575..4591)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4690..4707)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4861..5449)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4941..4960)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5571..6362,6363..6431))
                     /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     6194..6213
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6432..6523)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6565..6584
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6590..6609
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    6816..6849
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(7500..7665)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     7586..7610
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 gtgcgatcgc gtgcattcat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt
       61 ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca
      121 aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt
      181 gcttgagaag gttttgggac gctcgaagat cgcgtcagct gaaggtatac catgactgat
      241 tacgtacatg agatccagtc actcgggatc cgatctgcca attgaacata acatggtagt
      301 tacatatact agtaatatgg ttcggcacac attaaaagta taaaaactat ctgaattacg
      361 aattacatat attggtcata aaaatcaatc aatcatcgtg tgttttatat gtctcttatc
      421 taagtataag aatatccata gttaatattc acttacgcta ccttttaacc tgtaatcatt
      481 gtcaacagga tatgttaacg acccacattg ataaacgcta gtatttcttt ttcctcttct
      541 tattggccgg ctgtctctat actcccctat agtctgtttc ttttcgtttc gattgtccct
      601 atcagtgata gagatggcgc acatggtacg ctgtggtgct cgcggctggg aacgaaactc
      661 tgggagctgc gattggcact attctaaagt aacacattgt tttagagcta gaaatagcaa
      721 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      781 ctcgagccat atccaacttc caatttaatc tttctttttt aattttcact tatttgcgat
      841 acagaaagaa aaaagcgata gtaactattg aattttgttt ggatttggtt agattagata
      901 tggtttctct ttatatttac atgctaaaaa tgggcttcgt acgctgcagg tcgacaaccc
      961 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctagag atccgcaggc
     1021 taaccggaac ctgtattatt tagtttatgc tacgttaaat aaagaccttt cgttcacata
     1081 actgaatgtg taatggcctt gagatttcaa gcataccaag ttggtggaga cggggtcgtt
     1141 acaaaagact ctttaagcca agatttcctt gacagccttg gcgatagcat cgccaacctc
     1201 agtggtagag ttagaaccac caaggtcacc ggttctgaca cctgcatcca agacatttct
     1261 aacagcttct tcaagagccc taccttcttc aaccaaatcc aaggataact tcaacatcat
     1321 agctgcagat aagatggtag caattgggtt aaccttgttt gctggtaaat ctggggcaga
     1381 accatgacat ggttcgtaca aaccgaatgc cttgttagtg tcaggtaggg aagctagaga
     1441 tgcagaaggt aataaaccca aagaacctgg aataacagag gcttcatcgg agataatatc
     1501 accaaacatg ttgttggtaa taacaacacc gtttagctta gttggtgatt taaccaaaat
     1561 catagcagca gagtcgatca attggtgctg aacagttaat tgtgggaact cagtcttgat
     1621 ggtttcttca acagtctttc tccacaatct ggaagaggca agcacgttag ccttgtcaag
     1681 tgaccagatt ggtaatggtg ggttttgttg caatgccaag aaagcagcca ttcttgtaat
     1741 tctttgaact tcaggaacac tgtatttctc agagtcccaa gcaactccgt caccttcatc
     1801 ttcttttctt tcaccaaagt agataccacc aaccaattct ctaacgacga cgaaatcggt
     1861 accctttgca tattcaggct tcaaaggaga aagatctagt aaagaatcag aagcaaagtt
     1921 acatggtctt aagttggcgt atagacccaa ttcctttctg atcttcaata gaccttgttc
     1981 tggtctaact gcgcccgtac cccattttgg accaccaaca gcacctagta agacagcatc
     2041 ggctttctta gaggcttcta gagcttcatc tggtaaagga gtgccagtgg catcgatggc
     2101 agcacccccg atcaagtgat gttggaaatt gaacttaatt tctggacgga cttcagcaat
     2161 ggcattcaag accttaatag cttcgtcagt aacttcttta ccgacgtgat cacccggtag
     2221 gacaacgata ttcttagaca tggtataatc tgtgtagtgt gggatacttt ttacttcttc
     2281 aaataggtat caacaataaa aataaatcga agcaaatgta ggaatgcgtt aaagcagatg
     2341 tacttactta gagtacatag atatatttat ataattcaat atataaaagt atatgaagca
     2401 tctgatgttg aacctgtcat gactctaaac tggaatgggt agttatgctt ctgaaggttt
     2461 tccgttagaa aatgagtgca gggatcaggc ccggaaccgg ttttagcctg aaaggaaaag
     2521 aaaaaaatgc ggtgattact agcacgtgac tgcgctgaat tggaatccac cacaagtaca
     2581 gcgaatggca agagggaagg ggaatatatt gtgcctggcg gtagtctgct atatcaaatg
     2641 ttaaacataa ctaaatgcag tagtgaatag agtaatgtat ttctacatat gtgggtaggg
     2701 ccaatgggag cccgatgtct attgtacaga tattttcctt ttatagttaa tcaattcaac
     2761 ttggaaatca ttgagtcgat atctacttgg ataactggag ccgtggcagc gtttgaaatt
     2821 tgacgagtct ccttgttttg ttcgcgcatt ttcagtttat tcaaacggga gatggcaccg
     2881 aatccactga cttctctgtt caatttgaat ggtaatagga agaaagtaca tttcgagaaa
     2941 ttttcgccca acgtcgaaat ctcatggatg atatcctcga tacaaatgac accttcatca
     3001 cctaatttct cttcaacgat attgttgtca ttcaagatta tctcttttgg ttctttatct
     3061 tctggccttt gccacattat tctagatctc ttttggatta aagaacggat agatgcaagt
     3121 gaaggttgtc cgataacaat atatggtgat acaagtttca acaatgggta cacgtccttg
     3181 gtaagtttga tgaaaacacc agtgttcaac tcaacaagtc tcaaaagaga taacactttg
     3241 taagcctttg ctgggatctt cacagcgagc tctcgagaac ccttaatata acttcgtata
     3301 atgtatgcta tacgaagtta ttaggtgata tcagatccac tagtggccta tgcacccaat
     3361 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     3421 tgggaaaacc ctggcgttac ccctgcagga ctagtgctga ggcattaatt gtgtccgcgt
     3481 ttctaaggcc acttttcaat gaaacggata ttgatatgct agtaaaagga cgagctcaag
     3541 agcgaaaata taagtaaaga attcgagtgc acttgtctcc atgcagcaag atttcatatg
     3601 agtctttttt atctttttac tttttacatt acacgatatg cactttatga aaatttaacg
     3661 aggttggaag ccggataatc aaccaaaatc aggcacgaag gcacactcgt atatgcatgt
     3721 tgttgaaact ctgttacgct gaactaacaa tcacacatgt agaggtcacc gggaaaagtt
     3781 gcgaccccat ggaaggtcga tctcttcgtt tggctttgct tggctggcgg cattgcgctt
     3841 cttcgcttat acccgtctct tgacgctcga gctcgttcat tgagatacct ttattcttgc
     3901 acattttctg gcttttttcg ctactcgggt acatgtaatc atgcacacag aaggtgctgt
     3961 agggtgaaag ttcctttgtg ctgtcgtttg tttttaatgc caaactttcc ggtgatcaat
     4021 aaccacctca ttaagacctc agcgcggccg caaatttaaa taaaatgaag tgaagttcct
     4081 atactttcta gagaatagga acttctatag tgagtcgaat aagggcgaca caaaatttat
     4141 tctaaatgca taataaatac tgataacatc ttatagtttg tattatattt tgtattatcg
     4201 ttgacatgta taattttgat atcaaaaact gattttccct ttattatttt cgagatttat
     4261 tttcttaatt ctctttaaca aactagaaat attgtatata caaaaaatca taaataatag
     4321 atgaatagtt taattatagg tgttcatcaa tcgaaaaagc aacgtatctt atttaaagtg
     4381 cgttgctttt ttctcattta taaggttaaa taattctcat atatcaagca aagtgacagg
     4441 cgcccttaaa tattctgaca aatgctcttt ccctaaactc cccccataaa aaaacccgcc
     4501 gaagcgggtt tttacgttat ttgcggatta acgattactc gttatcagaa ccgcccaggg
     4561 ggcccgagct taagactggc cgtcgtttta caacacagaa agagtttgta gaaacgcaaa
     4621 aaggccatcc gtcaggggcc ttctgcttag tttgatgcct ggcagttccc tactctcgcc
     4681 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4741 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4801 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4861 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4921 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4981 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5041 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5101 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5161 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5221 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtgggc
     5281 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5341 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5401 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5461 gatcttttct acggggtctg acgctcagtg gaacgacgcg cgcgtaactc acgttaaggg
     5521 attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt aatgctctgc ttaccaatgc
     5581 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5641 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagcgctgcg
     5701 atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa ccagccagcc
     5761 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5821 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5881 atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5941 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6001 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6061 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6121 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6181 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6241 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6301 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6361 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6421 tgaatactca tattcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6481 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cagtgttaca
     6541 accaattaac caattctgaa cattatcgcg agcccattta tacctgaata tggctcataa
     6601 caccccttgt ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     6661 agaagtgaaa cgccgtagcg ccgatggtag tgtggggact ccccatgcga gagtagggaa
     6721 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgcccgggct
     6781 aattatgggg tgtcgccctt attcgactct atagtgaagt tcctattctc tagaaagtat
     6841 aggaacttct gaagtgggga tttaaatgcg gccgcgctga gggtttaatc gagatctttg
     6901 tgttcggtta cccggctcag atcctaactt cttcttttgg tatgtttatt cgtataagtt
     6961 actgttgtcc acaggcaata ctctgcagaa aattaaaacg gcattaatgc taggacaacc
     7021 agaattgtta ctactgtatg tgcgatagtt gataactgca acattatgcc cggtatattc
     7081 tcaaaaaacc ctattactgc atacgaagaa atcgcaagag aaatctttcg gtttggaaaa
     7141 gctcactgtg aggttccttg gagccaatag taatacagca caatccaagg aaaaatctgg
     7201 cctatatgca aggaaggaga gatagtcaaa agcattcttt cccctagaag ttggtgcata
     7261 tatggcatcg ttaaaacata ttacccccaa aatttcttct ctaaacgatg tgcttggcct
     7321 ttgttttggt ttttgatgtc ggtcgtttga ggccccttgc ggaaaatcga gatcgccgaa
     7381 tggcacgcga gggaagggaa ataaggttta aaggcactga aacaataggc aagaagtagg
     7441 cgagagccga catacgagac tattaagtcc tcagcgagct cgcatggaat gcgtgcgatg
     7501 agcgacctca tgctatacct gagaaagcaa cctgacctac aggaaagagt tactcaagaa
     7561 taagaatttt cgttttaaaa cctaagagtc actttaaaat ttgtatacac ttattttttt
     7621 tataacttat ttaataataa aaatcataaa tcataagaaa ttcgcttatt tagaagtgtc
     7681 aacaacgtat ctaccaacgg aatgc
//
