LOCUS       sequence-324998-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Divergent promoter expression cassette, P(HIS4) expression with 
            P(PGK1) normalizer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  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 6036)
  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: pNTI757 P(PGK1)-citrine P(HIS4)-mCherry
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     161..180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     414..431
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    548..569
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(584..589,590..607,608..614)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    622..638
                     /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     627..649
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     646..662
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     646..662
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        680..698
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     680..697
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1055..1074)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1324..1342
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             2569..3285
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3365..3384)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3366..3384)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3391..3408)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3391..3407)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3400..3422)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3545..3847
                     /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     3726..3745
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4196..4990
                     /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             4196..4990
                     /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(4250..4269)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4860..4879
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      5190..5199
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5196..5570
                     /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"
     rep_origin      join(5708..6036,1..260)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
       61 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      121 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      181 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      241 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      301 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      361 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      421 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      481 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      541 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      601 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      661 accatgatta cgccaagcta tttaggtgac actatagaat actcaagcta tgttcgccct
      721 tgtatcccct agggtaagtg actcgactgg cctcatagac agcggatcga gcgtgtctat
      781 ttgtacaatt catccatacc accagtagaa tgtctacctt cagctctttc atattgttcc
      841 acaatggtgt agtcctcgtt gtgggaggtg atgtccaact tgatgttgac gttgtaggcg
      901 ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcagc gtcgtagtgg
      961 ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg
     1021 tacattctct cggaggaggc ctcccagcac atggtcttct tctgcattac ggggccgtcg
     1081 gaggggaagt tggtgcctct caacttcacc ttgtagatga actcgccgtc ctgcagggag
     1141 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcactct ctcccacttg
     1201 aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag
     1261 gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggcca
     1321 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt agggtctgcc ctcgccctcg
     1381 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaatctcatg
     1441 aactccttga tgatggccat gttatcctcc tcgcccttgc tcaccatgga tcctattcag
     1501 aaaaaaaatt ttgtaaacta ttgtattact attacacagc gcagttgtgc tatgatattt
     1561 ttatgtatgt acaacacaca tcggaggtga atataacgtt ccatatctat tatatacaca
     1621 gtatactact gttcatagtc atatcctttt cttaccttct atatcgaatg actgataaaa
     1681 aaacgtgagt cactgtgcat gggtttagca attaattaac taatttaccg gagtcactat
     1741 tagagtcagt tcgactgcct aactagtata tgtaactacc atgttatgtt caattggcag
     1801 atcggacttc aactcaagac gcacagatat tataacatct gcataatagg catttgcaag
     1861 aattactcgt gagtaaggaa agagtgagga actatcgcat acctgcattt aaagatgccg
     1921 atttgggcgc gaatccttta ttttggcttc accctcatac tattatcagg gccagaaaaa
     1981 ggaagtgttt ccctccttct tgaattgatg ttaccctcat aaagcacgtg gcctcttatc
     2041 gagaaagaaa ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact gaaaaaaccc
     2101 agacacgctc gacttcctgt cttcctattg attgcagctt ccaatttcgt cacacaacaa
     2161 ggtcctagcg acggctcaca ggttttgtaa caagcaatcg aaggttctgg aatggcggga
     2221 aagggtttag taccacatgc tatgatgccc actgtgatct ccagagcaaa gttcgttcga
     2281 tcgtactgtt actctctctc tttcaaacag aattgtccga atcgtgtgac aacaacagcc
     2341 tgttctcaca cactcttttc ttctaaccaa gggggtggtt tagtttagta gaacctcgtg
     2401 aaacttacat ttacatatat ataaacttgc ataaattggt caatgcaaga aatacatatt
     2461 tggtcttttc taattcgtag tttttcaagt tcttagatgc tttctttttc tcttttttac
     2521 agatcatcaa ggaagtaatt atctactttt tacaacaaat gctagcaaat gtctaaaggt
     2581 gaagaattat tcactggtgt tgtcccaatt ttggttgaat tagatggtga tgttaatggt
     2641 cacaaatttt ctgtctccgg tgaaggtgaa ggtgatgcta cttacggtaa attgacctta
     2701 aaatttattt gtactactgg taaattgcca gttccatggc caaccttagt cactacttta
     2761 ggttatggtt tgatgtgttt tgctagatac ccagatcata tgaaacaaca tgactttttc
     2821 aagtctgcca tgccagaagg ttatgttcaa gaaagaacta tttttttcaa agatgacggt
     2881 aactacaaga ccagagctga agtcaagttt gaaggtgata ccttagttaa tagaatcgaa
     2941 ttaaaaggta ttgattttaa agaagatggt aacattttag gtcacaaatt ggaatacaac
     3001 tataactctc acaatgttta catcatggct gacaaacaaa agaatggtat caaagttaac
     3061 ttcaaaatta gacacaacat tgaagatggt tctgttcaat tagctgacca ttatcaacaa
     3121 aatactccaa ttggtgatgg tccagtcttg ttaccagaca accattactt atcctatcaa
     3181 tctgccttat ccaaagatcc aaacgaaaag agagaccaca tggtcttgtt agaatttgtt
     3241 actgctgctg gtattaccca tggtatggat gaattgtaca aatagtagcg acactagatg
     3301 gtctctgcga ccgttcctca ctgttagcac gtgaattcgg ataccatcta gagggcccaa
     3361 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     3421 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     3481 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctatacgt
     3541 acggcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     3601 acagagtgat attattgaca cgccggggcg acggatggtg atccccctgg ccagtgcacg
     3661 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     3721 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     3781 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     3841 aatataaatg tcaggcatga gattatcaaa aaggatcttc acctagatcc ttttcacgta
     3901 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
     3961 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
     4021 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
     4081 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     4141 ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgcatgat
     4201 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4261 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4321 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4381 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4441 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4501 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4561 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4621 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4681 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4741 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4801 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4861 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4921 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4981 gttcttctga attattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     5041 gtgcggtatt tcacaccgca tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5101 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5161 aaatgcttca ataatagcac gtgaggaggg ccaccatggc caagttgacc agtgccgttc
     5221 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     5281 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     5341 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     5401 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     5461 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     5521 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctaa
     5581 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5641 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5701 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5761 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5821 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5881 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5941 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6001 cggataaggc gcagcggtcg ggctgaacgg ggggtt
//
