LOCUS       sequence-471760-        5018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modified ADEPT-pCas9 with sfGFP under TtrB promoter for 
            tetrathionate (TTR) sensing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5018)
  AUTHORS   Son HI, Hamrick GS, Shende AR, Kim K, Yang K, Huang TJ, You L
  TITLE     Population-level amplification of gene regulation by programmable 
            gene transfer.
  JOURNAL   Nat Chem Biol. 2025 Jan 8. doi: 10.1038/s41589-024-01817-9.
  PUBMED    39779901
REFERENCE   2  (bases 1 to 5018)
  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: "Nat Chem 
            Biol. 2025 Jan 8. doi: 10.1038/s41589-024-01817-9."
COMMENT     Sequence Label: ADEPT-pTarget-tetR
FEATURES             Location/Qualifiers
     source          1..5018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            784..1074
                     /note="origin of transfer for the bacterial F plasmid 
                     (Frost et al., 1994)"
     primer_bind     1169..1186
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1239..1285)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1438..1455
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(1511..1528)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1544..1573)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(1594..1669)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(1690..1715,1716..1721,1722..1738,1739..1744,
                     1745..1763))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1720..1738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1745..1763
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      1781..1824
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             2034..2828
                     /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(2088..2107)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2698..2717
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2859..2953
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      complement(3064..3150)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3156..3782)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3687..3706
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     RBS             3789..3800
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      complement(4011..4038)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(4054..4125)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4137..4850)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     RBS             4857..4868
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctgtggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgaggtt caccttagcc attagagatt
      721 accttttttg aacataaaat aaagttaatt tcgaaacact aaaattcata atgcaaacag
      781 ggacgcaccg ctagcagcgc ccctagcggt atcctataaa aaaacacacc gcgccgctag
      841 cagcacccct aatataaaat aatgtttttt ataaaaatag tcagtaccac ccctacaaaa
      901 cggtgtcggc gcgttgttgt agccgcgccg acaccgcttt tttaaatatc ataaagagag
      961 taagagaaac taatttttca taacactcta tttataaaga aaaatcagca aaaacttgtt
     1021 tttgcatggg gtgtggtgct tttggtggtg agaaccacca acctgttgag cctttttgtg
     1081 gagtgggtta aattatttac gaataaagtc accagaggtg gcctttgagt gagctgatac
     1141 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cctgcagtct
     1201 agactcgagt aaggatccag ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     1261 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
     1321 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
     1381 accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat
     1441 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccaag ctggagaccg
     1501 tttaaactca atgatgatga tgatgatggt cgacggcgct attcagatcc tcttctgaga
     1561 tgagtttttg ttcgggccca agcttcaaaa aaagcaccga ctcggtgcca ctttttcaag
     1621 ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaacc gtgatacgcc
     1681 tatttttatg gtcagtgcgt cctgctgatg tgctcagtat ctctatcact gatagggatg
     1741 tcaatctcta tcactgatag ggaagatctt tagaattcca gaaatcatcc ttagcgaaag
     1801 ctaaggattt tttttatctg aaattctgcc tcgtgatacg cctattttta taggttaatg
     1861 tcatgataat aatggtttct tagacgtcag ggacgtcgga attgccagct ggggcgccct
     1921 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct
     1981 gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     2041 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     2101 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     2161 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     2221 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     2281 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     2341 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     2401 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     2461 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     2521 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     2581 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     2641 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     2701 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     2761 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     2821 tcttctgagc gggactctgg ggttcgagag ctcgcttgga ctcctgttga tagatccagt
     2881 aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt
     2941 tattggtgag aatccaagca ctagtaacaa cttatatcct gattctgtgg ataaccgtat
     3001 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgccctagg tctagggcgg
     3061 cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     3121 gtctttcgac tgagcctttc gttttatttg atgccttaag acccactttc acatttaagt
     3181 tgtttttcta atccgcatat gatcaattca aggccgaata agaaggctgg ctctgcacct
     3241 tggtgatcaa ataattcgat agcttgtcgt aataatggcg gcatactatc agtagtaggt
     3301 gtttcccttt cttctttagc gacttgatgc tcttgatctt ccaatacgca acctaaagta
     3361 aaatgcccca ctgcgctgag tgcatataat gcattctcta gtgaaaaacc ttgttggcat
     3421 aaaaaggcta attgattttc gagagtttca tactgttttt ctgtaggccg tgtacctaaa
     3481 tgtacttttg ctccatcgcg atgacttagt aaagcacatc taaaactttt agcgttatta
     3541 cgtaaaaaat cttgccagct ttccccttct aaagggcaaa agtgagtatg gtgcctatct
     3601 aacatctcaa tggctaaggc gtcgagcaaa gcccgcttat tttttacatg ccaatacaat
     3661 gtaggctgct ctacacctag cttctgggcg agtttacggg ttgttaaacc ttcgattccg
     3721 acctcattaa gcagctctaa tgcgctgtta atcactttac ttttatctaa acgagacatc
     3781 atctagtatt tctcctcttt acaggcattc tgtaaaaggc gaggcggcat aactatagtg
     3841 gttttccaca tatcaggcgt ggatttgatc tagcgcaaca aatattgact agctaaccag
     3901 ctcatttcag aatatttgcc agaaccgtta tgatgtcggc gcaaaaaaca ttatccagaa
     3961 cgggagtgcg tatggggctg acttcaggtg ctaccgtgga gacgtataaa cgcagaaagg
     4021 cccacccgaa ggtgagccag tgtgactcta gtagagagcg ttcaccgaca aacaacagat
     4081 aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggtcatttg
     4141 tacagttcat ccataccatg cgtgatgccc gctgcggtta cgaactccag cagaaccata
     4201 tgatcgcgtt tctcgttcgg atctttagac agaacgcttt gcgtgctcag atagtgattg
     4261 tctggcagca gaacaggacc atcaccgatt ggagtgtttt gctggtagtg atcagccagc
     4321 tgcacgctgc catcctccac gttgtggcga attttaaaat tcgctttaat gccatttttt
     4381 tgtttatcgg cggtgatgta aacattgtgg ctgttaaaat tgtattccag cttatggccc
     4441 aggatattgc cgtcctcttt aaagtcaatg cctttcagct caatgcggtt taccagggta
     4501 tcgccttcaa atttcacttc cgcacgcgtt ttgtacgtgc cgtcatcctt aaaggaaatc
     4561 gtgcgttcct gcacatagcc ttccggcatg gcggacttga agaagtcatg ctgcttcata
     4621 tggtccggat aacgagcaaa gcactgaaca ccataagtca gcgtcgttac cagagtcggc
     4681 caaggaaccg gcagtttacc agtagtacag atgaacttca gcgtcagttt accattagtt
     4741 gcgtcacctt caccctcgcc acgcacggaa aacttatgac cgttgacatc accatccagt
     4801 tccaccagaa tagggacgac accagtgaac agctcttcgc ctttacgcat ctagtatttc
     4861 tcctctttac aggcattctg taaaaggcga ggcggcataa ctatagtggt tttccacata
     4921 tcaggcgtgg atttgatcta gcgcaacaaa tattgacgcg ttgcgcagtt ttcgttccac
     4981 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
