LOCUS       sequence-471748-        4051 bp    DNA     circular SYN 07-MAY-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 4051)
  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 4051)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 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-Msp5
FEATURES             Location/Qualifiers
     source          1..4051
                     /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"
     primer_bind     complement(1828..1846)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     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(3020..3047)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3063..3134)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3146..3859)
                     /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             3866..3877
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3886..3908)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3897..3913
                     /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(3921..3927,3928..3945,3946..3951))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3966..3987
                     /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 gtgatactcc
     1681 taatgttatg 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 cttatatcgt atggggctga cttcaggtgc
     3001 taccgtggag acgtataaac gcagaaaggc ccacccgaag gtgagccagt gtgactctag
     3061 tagagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc
     3121 tttcgtttta tttgatgcct ggtcatttgt acagttcatc cataccatgc gtgatgcccg
     3181 ctgcggttac gaactccagc agaaccatat gatcgcgttt ctcgttcgga tctttagaca
     3241 gaacgctttg cgtgctcaga tagtgattgt ctggcagcag aacaggacca tcaccgattg
     3301 gagtgttttg ctggtagtga tcagccagct gcacgctgcc atcctccacg ttgtggcgaa
     3361 ttttaaaatt cgctttaatg ccattttttt gtttatcggc ggtgatgtaa acattgtggc
     3421 tgttaaaatt gtattccagc ttatggccca ggatattgcc gtcctcttta aagtcaatgc
     3481 ctttcagctc aatgcggttt accagggtat cgccttcaaa tttcacttcc gcacgcgttt
     3541 tgtacgtgcc gtcatcctta aaggaaatcg tgcgttcctg cacatagcct tccggcatgg
     3601 cggacttgaa gaagtcatgc tgcttcatat ggtccggata acgagcaaag cactgaacac
     3661 cataagtcag cgtcgttacc agagtcggcc aaggaaccgg cagtttacca gtagtacaga
     3721 tgaacttcag cgtcagttta ccattagttg cgtcaccttc accctcgcca cgcacggaaa
     3781 acttatgacc gttgacatca ccatccagtt ccaccagaat agggacgaca ccagtgaaca
     3841 gctcttcgcc tttacgcatc tagtatttct cctctttagc tgtttcctgt gtgaaattgt
     3901 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3961 gcctaatgag tgagctaact cacattaatt gcgttgcgca gttttcgttc cactgagcgt
     4021 cagaccccgt agaaaagatc aaaggatctt c
//
