LOCUS       sequence-357453-        7431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET28a-NLS-M-MLV RT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7431)
  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 7431)
  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 
            Biotechnol. 2022 Apr 4. pii: 10.1038/s41587-022-01255-9. doi: 
            10.1038/s41587-022-01255-9."
COMMENT     Sequence Label: pET28a-NLS-M-MLV-RT
FEATURES             Location/Qualifiers
     source          1..7431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1295..1314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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)."
     RBS             1456..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1497..1514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1524..1541
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1551..1571
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3669..3689
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3693..3710
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3763..3781)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3777..3824
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3861..4316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3948..3967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4158..4179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4408..5223)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4408..5223)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5131..5150
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5215..5234
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5345..5933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5834..5853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6087..6104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6119..6258
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6184..6203)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6344..6366
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6360..6551)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7360..7431,1..1011))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     1501 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatggcc ccaaagaaga
     1561 agcggaaggt cggtatccac ggagtcccag cagccaccct aaatatagaa gatgagtatc
     1621 ggctacatga gacctcaaaa gagccagatg tttctctagg gtccacatgg ctgtctgatt
     1681 ttcctcaggc ctgggcggaa accgggggca tgggactggc agttcgccaa gctcctctga
     1741 tcatacctct gaaagcaacc tctacccccg tgtccataaa acaatacccc atgtcacaag
     1801 aagccagact ggggatcaag ccccacatac agagactgtt ggaccaggga atactggtac
     1861 cctgccagtc cccctggaac acgcccctgc tacccgttaa gaaaccaggg actaatgatt
     1921 ataggcctgt ccaggatctg agagaagtca acaagcgggt ggaagacatc caccccaccg
     1981 tgcccaaccc ttacaacctc ttgagcgggc tcccaccgtc ccaccagtgg tacactgtgc
     2041 ttgatttaaa ggatgccttt ttctgcctga gactccaccc caccagtcag cctctcttcg
     2101 cctttgagtg gagagatcca gagatgggaa tctcaggaca attgacctgg accagactcc
     2161 cacagggttt caaaaacagt cccaccctgt ttaatgaggc actgcacaga gacctagcag
     2221 acttccggat ccagcaccca gacttgatcc tgctacagta cgtggatgac ttactgctgg
     2281 ccgccacttc tgagctagac tgccaacaag gtactcgggc cctgttacaa accctaggga
     2341 acctcgggta tcgggcctcg gccaagaaag cccaaatttg ccagaaacag gtcaagtatc
     2401 tggggtatct tctaaaagag ggtcagagat ggctgactga ggccagaaaa gagactgtga
     2461 tggggcagcc tactccgaag acccctcgac aactaaggga gttcctaggg aaggcaggct
     2521 tctgtcgcct cttcatccct gggtttgcag aaatggcagc ccccttgtac cctctcacca
     2581 aaccggggac tctgtttaat tggggcccag accaacaaaa ggcctatcaa gaaatcaagc
     2641 aagctcttct aactgcccca gccctggggt tgccagattt gactaagccc tttgaactct
     2701 ttgtcgacga gaagcagggc tacgccaaag gtgtcctaac gcaaaaactg ggaccttggc
     2761 gtcggccggt ggcctacctg tccaaaaagc tagacccagt agcagctggg tggccccctt
     2821 gcctacggat ggtagcagcc attgccgtac tgacaaagga tgcaggcaag ctaaccatgg
     2881 gacagccact agtcattctg gccccccatg cagtagaggc actagtcaaa caaccccccg
     2941 accgctggct ttccaacgcc cggatgactc actatcaggc cttgcttttg gacacggacc
     3001 gggtccagtt cggaccggtg gtagccctga acccggctac gctgctccca ctgcctgagg
     3061 aagggctgca acacaactgc cttgatatcc tggccgaagc ccacggaacc cgacccgacc
     3121 taacggacca gccgctccca gacgccgacc acacctggta tacggatgga agcagtctct
     3181 tacaagaggg acagcgtaag gcgggagctg cggtgaccac cgagaccgag gtaatctggg
     3241 ctaaagccct gccagccggg acatccgctc agcgggctga actgatagca ctcacccagg
     3301 ccctaaagat ggcagaaggt aagaagctaa atgtttatac tgatagccgt tatgcttttg
     3361 ctactgccca tatccatgga gaaatataca gaaggcgtgg gtggctcaca tcagaaggca
     3421 aagagatcaa aaataaagac gagatcttgg ccctactaaa agccctcttt ctgcccaaaa
     3481 gacttagcat aatccattgt ccaggacatc aaaagggaca cagcgccgag gctagaggca
     3541 accggatggc tgaccaagcg gcccgaaagg cagccatcac agagactcca gacacctcta
     3601 ccctcctcat cgagaatagc tcgccgagtg gaggctccaa acgcaccgct gacggcagtg
     3661 agtttgaacc aaagaagaag cgcaaagtcg agcaccacca ccaccaccac tgagatccgg
     3721 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     3781 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     3841 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3901 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3961 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4021 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4081 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4141 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4201 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4261 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     4321 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4381 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     4441 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     4501 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     4561 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     4621 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     4681 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     4741 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     4801 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     4861 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     4921 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     4981 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     5041 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     5101 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     5161 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     5221 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     5281 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5341 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5401 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5461 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     5521 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5581 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5641 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5701 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5761 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5821 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5881 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5941 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6001 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6061 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6121 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     6181 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     6241 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6301 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6361 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     6421 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     6481 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     6541 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     6601 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     6661 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     6721 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     6781 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     6841 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     6901 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     6961 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     7021 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     7081 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     7141 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     7201 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     7261 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     7321 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     7381 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag a
//
