LOCUS       sequence-481789-        7662 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  For circular RNA-mediated inverse prime editor using MCP-Rep-X-M 
            MLV-RT∆RNase H in HEK294T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7662)
  TITLE     Circular RNA-mediated inverse prime editing in human cells
REFERENCE   2  (bases 1 to 7662)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: MCP-Rep-X-RT∆R
FEATURES             Location/Qualifiers
     source          1..7662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             694..714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3259..3279
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4897..4917
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4926..4943
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4966..4983)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4972..5196
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5267..5283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5267..5283)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5280..5302)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5291..5307
                     /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(5315..5321,5322..5339,5340..5345))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5360..5381
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5498..5515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5669..6257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5749..5768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6428..7219,7220..7288))
                     /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     7051..7070
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7289..7393)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7472..7491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcaagctt
      721 gcgtccaact tcacccaatt tgtactggtg gacaatggtg gaacaggaga cgtgaccgtg
      781 gccccttcta acttcgccaa tggaatcgca gagtggattt cctcaaactc ccggtcacag
      841 gcatacaaag tgacgtgttc agtgaggcag tcctccgctc agaaccgcaa atacaccatt
      901 aaggttgaag tgccaaaagg cgcctggagg tcctacttga atatggagct taccattcca
      961 atttttgcca caaatagcga ttgtgagctg attgtgaagg ccatgcaggg cctcctgaag
     1021 gacgggaacc ccattccgag cgccatagca gcaaatagcg gaatttattc tagactgcag
     1081 tctggaggat ctagcggagg atcctctggc agcgagacac caggaacaag cgagtcagca
     1141 acaccagaga gcagtggcgg cagcagcggc ggcagcgagc tcatgagact gaaccccggc
     1201 caacagcagg ctgtggaatt tgtgaccggc cccctgctgg tgctggctgg agctggaagc
     1261 ggaaaaacaa gagtgattac aaacaagatc gcccacctga tcagaggcag cggctatcaa
     1321 gctagacaca tcgccgctgt gaccttcacc aacaaggccg ccagagagat gaaggagaga
     1381 gtgggccaaa ccctgggcag aaaggaggcc agaggactga tgattagcac cttccacacc
     1441 ctgggcctgg acatcattaa gagagagtac gccgccctgg gcatgaaggc caactttagc
     1501 ctgtttgacg acaccgacca gctggccctt ctgaaagaac tgacagaagg cctgatcgag
     1561 gacgacaagg tgctgctgca acagctgatt agcaccatca gcaactggaa gaacgacctg
     1621 aagaccccca gccaggccgc tgcaagcgcc attggagaaa gagatagaat cttcgcccac
     1681 gtgtacggcc tgtacgacgc ccacctgaaa gcctgcaatg tgctggattt cgacgacctg
     1741 atcctgctgc ccaccctgct gctgcaaaga aacgaagagg tgagaaagag atggcagaac
     1801 aagatcagat acctgctggt ggacgagtac caggacacca acaccagcca gtacgagctg
     1861 gtgaagctgc tggtgggcag cagagctaga ttcaccgtgg tgggcgatga cgaccagagc
     1921 atttatagct ggagaggcgc cagaccccag aatctggttc tgctgtctca agactttccc
     1981 gccctgaaag tgattaagct ggagcagaac tacagaagca gcggcagaat cctgaaggcc
     2041 gccaatattc tgatcgccaa caacccccat gtgttcgaga agagactgtt cagcgagctg
     2101 ggctacggcg ccgaactgaa agtgctgagc gctaacaatg aagagcacga ggccgagaga
     2161 gtgaccggcg aactgattgc tcatcacttc gtgaacaaaa cccagtacaa ggactacgcc
     2221 atcctgtaca gaggcaacca ccagagcaga gtgttcgaga agttcctgat gcagaacaga
     2281 atcccctaca agatcagcgg cggcaccagc ttttttagca gacccgaaat caaggacctg
     2341 ctggcctacc tgagagtgct gaccaatccc gacgatgact gcgcctttct gagaatcgtg
     2401 aacaccccca agagagagat cggccccgcc acactgaaaa agctgggcga atgggctatg
     2461 acaagaaaca agagcatgtt caccgccagc ttcgacatgg gcctgagcca aacactgagc
     2521 ggcagaggat atgaagccct gacaagattc acccactggc tggccgagat tcagagactg
     2581 gccgaaagag agcccatcgc cgctgttagg gatctgattc acggaatgga ttacgagagc
     2641 tggctgtacg agacaagccc cagccctaaa gctgctgaga tgagaatgaa gaacgtgaac
     2701 cagctgttca gctggatgac cgagatgctg gagggcagcg aactggacga acctatgaca
     2761 ctgacacagg tggtgaccag attcaccctg agagacatga tggagagagg cgagagcgag
     2821 gaggagctgg atcaagtgca actgatgaca ctgcacgcca gcaagggcct ggagtttccc
     2881 tatgtgtata tggtgggcat ggaggagggc ttcctgcccc atcaaagcag cattgatgag
     2941 gacaacatcg acgaggagag aagactggcc tacgtgggca ttaccagagc ccagaaagaa
     3001 ctgacattca ccctggccaa ggagagaaga cagtacggcg agctggtcag acccgagcct
     3061 agcagatttc tgctggagct gccccaggac gacctgattt gggaacaaga aagaaaggtg
     3121 gtgagcgccg aggagagaat gcagaagggc caaagccatc tggccaatct gaaggctatg
     3181 atggccgcca aaagaggcaa gagcggcggc tctagcggag gaagcaaaag aacagctgac
     3241 ggcagcgagt tcgagagccc caaaaagaaa agaaaggtga gcggcggcag cagcggcgga
     3301 tctcccggga gcaccctaaa tatagaagat gagtatcggc tacatgagac ctcaaaagag
     3361 ccagatgttt ctctagggtc cacatggctg tctgattttc ctcaggcctg ggcggaaacc
     3421 gggggcatgg gactggcagt tcgccaagct cctctgatca tacctctgaa agcaacctct
     3481 acccccgtgt ccataaaaca ataccccatg tcacaagaag ccagactggg gatcaagccc
     3541 cacatacaga gactgttgga ccagggaata ctggtaccct gccagtcccc ctggaacacg
     3601 cccctgctac ccgttaagaa accagggact aatgattata ggcctgtcca ggatctgaga
     3661 gaagtcaaca agcgggtgga agacatccac cccaccgtgc ccaaccctta caacctcttg
     3721 agcgggctcc caccgtccca ccagtggtac actgtgcttg atttaaagga tgcctttttc
     3781 tgcctgagac tccaccccac cagtcagcct ctcttcgcct ttgagtggag agatccagag
     3841 atgggaatct caggacaatt gacctggacc agactcccac agggtttcaa aaacagtccc
     3901 accctgttta atgaggcact gcacagagac ctagcagact tccggatcca gcacccagac
     3961 ttgatcctgc tacagtacgt ggatgactta ctgctggccg ccacttctga gctagactgc
     4021 caacaaggta ctcgggccct gttacaaacc ctagggaacc tcgggtatcg ggcctcggcc
     4081 aagaaagccc aaatttgcca gaaacaggtc aagtatctgg ggtatcttct aaaagagggt
     4141 cagagatggc tgactgaggc cagaaaagag actgtgatgg ggcagcctac tccgaagacc
     4201 cctcgacaac taagggagtt cctagggaag gcaggcttct gtcgcctctt catccctggg
     4261 tttgcagaaa tggcagcccc cctgtaccct ctcaccaaac cggggactct gtttaattgg
     4321 ggcccagacc aacaaaaggc ctatcaagaa atcaagcaag ctcttctaac tgccccagcc
     4381 ctggggttgc cagatttgac taagcccttt gaactctttg tcgacgagaa gcagggctac
     4441 gccaaaggtg tcctaacgca aaaactggga ccttggcgtc ggccggtggc ctacctgtcc
     4501 aaaaagctag acccagtagc agctgggtgg cccccttgcc tacggatggt agcagccatt
     4561 gccgtactga caaaggatgc aggcaagcta accatgggac agccactagt cattctggcc
     4621 ccccatgcag tagaggcact agtcaaacaa ccccccgacc gctggctttc caacgcccgg
     4681 atgactcact atcaggcctt gcttttggac acggaccggg tccagttcgg accggtggta
     4741 gccctgaacc cggctacgct gctcccactg cctgaggaag ggctgcaaca caactgcctt
     4801 gatatcctgg ccgaagccca cggaacccga cccgacctaa cggaccagcc gctctctggc
     4861 ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa
     4921 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     4981 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5041 ccactcccac tgtcctttcc taataaaatg agaaaattgc atcgcattgt ctgagtaggt
     5101 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5161 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5221 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5281 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5341 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5401 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5461 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5521 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5581 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5641 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5701 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5761 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5821 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5881 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5941 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6001 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6061 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6121 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6181 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6241 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga
     6301 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6361 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6421 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6481 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6541 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6601 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6661 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6721 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6781 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6841 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6901 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6961 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7021 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7081 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7141 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7201 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7261 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7321 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7381 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7441 gatctcccga tcccctaggg tctactctca gtacaatctg ctctgatgcc gcatagttaa
     7501 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     7561 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     7621 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
