LOCUS       sequence-471770-        4050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sfGFP under lac promoter. Expresses a self-targeting 
            mismatched guide RNA as part of the ADEPT system. Carries oriT and 
            kanamycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4050)
  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 4050)
  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-Msp4
FEATURES             Location/Qualifiers
     source          1..4050
                     /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(1689..1714,1715..1720,1721..1737,1738..1743,
                     1744..1762))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1719..1737
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1744..1762
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      1780..1823
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(1827..1845)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             2033..2827
                     /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(2087..2106)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2697..2716
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2858..2952
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      complement(3019..3046)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3062..3133)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3145..3858)
                     /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             3865..3876
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3885..3907)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3896..3912
                     /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(3920..3926,3927..3944,3945..3950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3965..3986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
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 gtgatacgct
     1681 aattttaagg tcagtgcgtc ctgctgatgt gctcagtatc tctatcactg atagggatgt
     1741 caatctctat cactgatagg gaagatcttt agaattccag aaatcatcct tagcgaaagc
     1801 taaggatttt ttttatctga aattctgcct cgtgatacgc ctatttttat aggttaatgt
     1861 catgataata atggtttctt agacgtcagg gacgtcggaa ttgccagctg gggcgccctc
     1921 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     1981 atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     2041 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2101 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     2161 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     2221 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     2281 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     2341 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2401 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     2461 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     2521 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     2581 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     2641 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     2701 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     2761 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     2821 cttctgagcg ggactctggg gttcgagagc tcgcttggac tcctgttgat agatccagta
     2881 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     2941 attggtgaga atccaagcac tagtaacaac ttatatcgta tggggctgac ttcaggtgct
     3001 accgtggaga cgtataaacg cagaaaggcc cacccgaagg tgagccagtg tgactctagt
     3061 agagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
     3121 ttcgttttat ttgatgcctg gtcatttgta cagttcatcc ataccatgcg tgatgcccgc
     3181 tgcggttacg aactccagca gaaccatatg atcgcgtttc tcgttcggat ctttagacag
     3241 aacgctttgc gtgctcagat agtgattgtc tggcagcaga acaggaccat caccgattgg
     3301 agtgttttgc tggtagtgat cagccagctg cacgctgcca tcctccacgt tgtggcgaat
     3361 tttaaaattc gctttaatgc catttttttg tttatcggcg gtgatgtaaa cattgtggct
     3421 gttaaaattg tattccagct tatggcccag gatattgccg tcctctttaa agtcaatgcc
     3481 tttcagctca atgcggttta ccagggtatc gccttcaaat ttcacttccg cacgcgtttt
     3541 gtacgtgccg tcatccttaa aggaaatcgt gcgttcctgc acatagcctt ccggcatggc
     3601 ggacttgaag aagtcatgct gcttcatatg gtccggataa cgagcaaagc actgaacacc
     3661 ataagtcagc gtcgttacca gagtcggcca aggaaccggc agtttaccag tagtacagat
     3721 gaacttcagc gtcagtttac cattagttgc gtcaccttca ccctcgccac gcacggaaaa
     3781 cttatgaccg ttgacatcac catccagttc caccagaata gggacgacac cagtgaacag
     3841 ctcttcgcct ttacgcatct agtatttctc ctctttagct gtttcctgtg tgaaattgtt
     3901 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3961 cctaatgagt gagctaactc acattaattg cgttgcgcag ttttcgttcc actgagcgtc
     4021 agaccccgta gaaaagatca aaggatcttc
//
