LOCUS       sequence-357443-        4536 bp    DNA     circular SYN 14-APR-2025
DEFINITION  petRNA cloning plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4536)
  AUTHORS   Liu B, Dong X, Cheng H, Zheng C, Chen Z, Rodriguez TC, Liang SQ, Xue
            W, Sontheimer EJ
  TITLE     A split prime editor with untethered reverse transcriptase and 
            circular RNA template.
  JOURNAL   Nat Biotechnol. 2022 Apr 4. pii: 10.1038/s41587-022-01255-9. doi: 
            10.1038/s41587-022-01255-9.
  PUBMED    35379962
REFERENCE   2  (bases 1 to 4536)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2022 Apr 4. pii: 10.1038/s41587-022-01255-9. doi: 
            10.1038/s41587-022-01255-9."
COMMENT     Sequence Label: U6-petRNA
FEATURES             Location/Qualifiers
     source          1..4536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..31
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    148..169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(184..189,190..207,208..214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    222..238
                     /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     227..249
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     246..262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     246..262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     281..301
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        283..301
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        348..596
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     348..368
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     519..538
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        716..734
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     CDS             complement(866..1660)
                     /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(977..996)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1587..1606
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(1963..1979)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1964..1980)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2004..2023)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2005..2023)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2033..2050)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2033..2049)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2042..2064)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2191..2646
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2278..2297)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2488..2509
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2672..2776
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2777..2845,2846..3637)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2995..3014)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3808..4396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4297..4316
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
       61 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      121 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      181 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      241 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      301 ggaacaaaag ctggagctcc accgcggtgg cggccgcccc cttcaccgag ggcctatttc
      361 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
      421 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
      481 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
      541 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtgc
      601 tcgcttcggc agcacatata ctagtcgacg ggccgcactc gccggtccca agcccggata
      661 aaatgggagg gggcgggaaa ccgcctaacc atgccgagtg cggccgcaac taagcacatg
      721 aggatcaccc atgtgcgtgt gcaggtgagt gatccaaacg cccggcggca accgagcgtt
      781 ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc
      841 gagctctcga accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga
      901 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
      961 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
     1021 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
     1081 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga
     1141 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
     1201 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
     1261 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
     1321 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
     1381 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
     1441 ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg
     1501 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
     1561 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
     1621 cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg
     1681 tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc
     1741 agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccgacg
     1801 gatcagctag cagaacctgc tgacaaatta acagtggccg cggtcggcgt ggactgtaga
     1861 acactgccaa tgccggtccc aagcccggat aaaagtggag ggtacagtcc acgctctaga
     1921 gcggacttcg gtccgctttt ttgaattcga tatcaagctt atcgataccg tcgacctcga
     1981 gggggggccc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     2041 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2101 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2161 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     2221 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2281 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2341 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2401 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2461 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2521 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2581 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     2641 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2701 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2761 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     2821 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     2881 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     2941 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3001 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3061 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3121 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3181 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3241 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3301 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3361 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3421 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3481 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3541 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3601 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3661 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3721 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3781 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3841 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3901 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     3961 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4021 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4081 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4141 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4201 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4261 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4321 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4381 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4441 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4501 cgcctttgag tgagctgata ccgctcgccg cagccg
//
