LOCUS       sequence-476072-        4096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sfGFP under lac promoter. Expresses a single guide RNA 
            (under Rha promoter), which targets a noncoding region of the 
            plasmid as part of the ADEPT system. Carries oriT and kan 
            resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4096)
  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 4096)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..4096
                     /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(1690..1808)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     terminator      1826..1869
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(1873..1891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             2079..2873
                     /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(2133..2152)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2743..2762
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2904..2998
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      complement(3065..3092)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3108..3179)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3191..3904)
                     /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             3911..3922
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3931..3953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3942..3958
                     /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(3966..3972,3973..3990,3991..3996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4011..4032
                     /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 gtgagacgcc
     1681 tatttttatt acgaccagtc taaaaagcgc ctgaattcgc gaccttctcg ttactgacag
     1741 gaaaatgggc cattggcaac cagggaaaga tgaacgtgat gatgttcaca atttgctgaa
     1801 ttgtggtgag atctttagaa ttccagaaat catccttagc gaaagctaag gatttttttt
     1861 atctgaaatt ctgcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     1921 tttcttagac gtcagggacg tcggaattgc cagctggggc gccctctggt aaggttggga
     1981 agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat
     2041 caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2101 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2161 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2221 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2281 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2341 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2401 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2461 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2521 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2581 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2641 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     2701 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     2761 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     2821 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     2881 tctggggttc gagagctcgc ttggactcct gttgatagat ccagtaatga cctcagaact
     2941 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     3001 aagcactagt aacaacttat atcgtatggg gctgacttca ggtgctaccg tggagacgta
     3061 taaacgcaga aaggcccacc cgaaggtgag ccagtgtgac tctagtagag agcgttcacc
     3121 gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga
     3181 tgcctggtca tttgtacagt tcatccatac catgcgtgat gcccgctgcg gttacgaact
     3241 ccagcagaac catatgatcg cgtttctcgt tcggatcttt agacagaacg ctttgcgtgc
     3301 tcagatagtg attgtctggc agcagaacag gaccatcacc gattggagtg ttttgctggt
     3361 agtgatcagc cagctgcacg ctgccatcct ccacgttgtg gcgaatttta aaattcgctt
     3421 taatgccatt tttttgttta tcggcggtga tgtaaacatt gtggctgtta aaattgtatt
     3481 ccagcttatg gcccaggata ttgccgtcct ctttaaagtc aatgcctttc agctcaatgc
     3541 ggtttaccag ggtatcgcct tcaaatttca cttccgcacg cgttttgtac gtgccgtcat
     3601 ccttaaagga aatcgtgcgt tcctgcacat agccttccgg catggcggac ttgaagaagt
     3661 catgctgctt catatggtcc ggataacgag caaagcactg aacaccataa gtcagcgtcg
     3721 ttaccagagt cggccaagga accggcagtt taccagtagt acagatgaac ttcagcgtca
     3781 gtttaccatt agttgcgtca ccttcaccct cgccacgcac ggaaaactta tgaccgttga
     3841 catcaccatc cagttccacc agaataggga cgacaccagt gaacagctct tcgcctttac
     3901 gcatctagta tttctcctct ttagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4021 taactcacat taattgcgtt gcgcagtttt cgttccactg agcgtcagac cccgtagaaa
     4081 agatcaaagg atcttc
//
