LOCUS       sequence-273895-        6881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  phage cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6881)
  AUTHORS   Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, 
            Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR
  TITLE     Phage-assisted evolution of an adenine base editor with improved Cas
            domain compatibility and activity.
  JOURNAL   Nat Biotechnol. 2020 Jul;38(7):883-891. doi: 
            10.1038/s41587-020-0453-z. Epub 2020 Mar 16.
  PUBMED    32433547
REFERENCE   2  (bases 1 to 6881)
  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"; doi: 
            "10.1038/s41587-020-0453-z"; journalName: "Nat Biotechnol"; date: 
            "2020-07"; volume: "38"; issue: "7"; pages: "883-891"
COMMENT     Sequence Label: pBT114
FEATURES             Location/Qualifiers
     source          1..6881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      983..1437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1070..1089)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1279..1300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    1489..1507
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1514..1532
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1550..1561
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1568..2245
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2263..2334
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2350..2377
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2476..3291)
                     /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(2476..3291)
                     /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     3199..3218
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3283..3302
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3359..4309)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4357..4579
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5232..5570
                     /codon_start=1
                     /gene="VI"
                     /product="pVI"
                     /label=M13 gene VI
                     /note="minor coat protein"
                     /translation="MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLA
                     LIIGLNSILVGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKTAIFIFDVKQKI
                     VSYLDWDK"
     CDS             5572..6618
                     /codon_start=1
                     /gene="I"
                     /product="pI"
                     /label=M13 gene I
                     /note="phage assembly"
                     /translation="MAVYFVTGKLGSGKTLVSVGKIQDKIVAGCKIATNLDLRLQNLPQ
                     VGRFAKTPRVLRIPDKPSISDLLAIGRGNDSYDENKNGLLVLDECGTWFNTRSWNDKER
                     QPIIDWFLHARKLGWDIIFLVQDLSIVDKQARSALAEHVVYCRRLDRITLPFVGTLYSL
                     ITGSKMPLPKLHVGVVKYGDSQLSPTVERWLYTGKNLYNAYDTKQAFSSNYDSGVYSYL
                     TPYLSHGRYFKPLNLGQKMKLTKIYLKKFSRVLCLAIGFASAFTYSYITQPKPEVKKVV
                     SQTYDFDKFTIDSSHRLNLSYRYVFKDSKGKLINSDDLQKQGYSLTYIDLCTVSIKKGN
                     SNEIVKCN"
     CDS             join(6596..6881,1..995)
                     /codon_start=1
                     /gene="IV"
                     /product="pIV"
                     /label=M13 gene IV
                     /note="phage assembly"
                     /translation="MKLLNVINFVFLMFVSSSSFAQVIEMNNSPLRDFITWYSKQSGES
                     VIVSPDVKGTVTVYSSDVKPENLRNFFISVLRANNFDMVGSNPSIIQKYNPNNQDYIDE
                     LPSPDNQEYDDNSAPSGGFFVPQNDNVTQTFKINNVRAKDLIRVVELFVKSNTSKSSNV
                     LSIDGSNLLVVSAPKDILDNLSQFLSTVDLPTDQVLIEGLIFEVQQGDALDFSFAAGSQ
                     RGTVAGGVNTDRLTSVLSSAGGSFGIFNGDVLGLSVRALKTNSHSKILSVPRILTLSGQ
                     KGSISIGQNVPFITGRVTGESANVNNPFQTIERQNVGISMSVFPVAMAGGNIVLDITSK
                     ADSLSSSTQASDVITNQRSIATTVNLRDGQTLLLGGLTDYKNTSQDSGVPFLSKIPLIG
                     LLFSSRSDSNEESTLYVLVKATIVRAL"
ORIGIN
        1 caaacaatca ggattatatt gatgaattgc catcacctga taatcaggaa tatgatgata
       61 attccgctcc ttctggtggt ttctttgtcc cgcaaaatga taatgttact caaactttta
      121 aaattaataa cgttcgggca aaggatttaa tacgagttgt cgaattgttt gtaaagtcta
      181 atacttctaa atcctcaaat gtattatcta ttgacggatc taatctatta gttgttagtg
      241 ctcctaaaga tattttagat aacctttctc aattcctttc aactgttgat ttgccaactg
      301 accaggtatt gattgagggt ttgatatttg aggttcagca aggtgatgct ttagattttt
      361 catttgctgc tggctctcag cgtggcactg ttgcaggcgg agttaatact gaccgcctca
      421 cctctgtttt atcttctgct ggtggttcgt tcggtatttt taatggcgat gttttagggc
      481 tatcagttcg cgcattaaag actaatagcc attcaaaaat attgtctgtg ccacgtattc
      541 ttacgctttc aggtcagaag ggttctatct ctattggcca gaatgtccct tttattactg
      601 gtcgtgtaac tggtgaatct gccaatgtaa ataatccatt tcagacgatt gagcgtcaaa
      661 atgtaggtat ttccatgagc gtttttcctg ttgcaatggc tggcggtaat attgttctgg
      721 atattaccag caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta
      781 accaaagaag tattgctaca acggttaatt tgcgtgatgg acagactctt ctactcggtg
      841 gcctcactga ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc
      901 ctttaatcgg cctcctgttt agctcccgct ctgattctaa cgaggagagc acgttatacg
      961 tgctcgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1021 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1081 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagcgct aaatcggggg
     1141 cccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     1201 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1261 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1321 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1381 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa
     1441 tatttgctta tacaatcttc ctgtttttgg ggcttttctt agaagagctc cctatcagtg
     1501 atagagattg acatccctat cagtgataga gatactgagc acgaatctga aagaggagaa
     1561 aggatctatg gcgagtagcg aagacgttat caaagagttc atgcgtttca aagttcgtat
     1621 ggaaggttcc gttaacggtc acgagttcga aatcgaaggt gaaggtgaag gtcgtccgta
     1681 cgaaggtacc cagaccgcta aactgaaagt taccaaaggt ggtccgctgc cgttcgcttg
     1741 ggacatcctg tccccgcagt tccagtacgg ttccaaagct tacgttaaac acccggctga
     1801 catcccggac tacctgaaac tgtccttccc ggaaggtttc aaatgggaac gtgttatgaa
     1861 cttcgaagac ggtggtgttg ttaccgttac ccaggactcc tccctgcaag acggtgagtt
     1921 catctacaaa gttaaactgc gtggtaccaa cttcccgtcc gacggtccgg ttatgcagaa
     1981 aaaaaccatg ggttgggaag cttccaccga acgtatgtac ccggaagacg gtgctctgaa
     2041 aggtgaaatc aaaatgcgtc tgaaactgaa agacggtggt cactacgacg ctgaagttaa
     2101 aaccacctac atggctaaaa aaccggttca gctgccgggt gcttacaaaa ccgacatcaa
     2161 actggacatc acctcccaca acgaagacta caccatcgtt gaacagtacg aacgtgctga
     2221 aggtcgtcac tccaccggtg cttaataagg atctccaggc atcaaataaa acgaaaggct
     2281 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     2341 agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacaggtgg cacttttcgg
     2401 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2461 ctcataaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     2521 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2581 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2641 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2701 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2761 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2821 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2881 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2941 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     3001 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     3061 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     3121 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     3181 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     3241 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     3301 tgtattactg tttatgtaag cagacagttt tattgttcat gaaggccaaa taggccgttc
     3361 agatccttcc gtatttagcc agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc
     3421 atgagaacga accattgaga tcatgcttac tttgcatgtc actcaaaaat tttgcctcaa
     3481 aactggtgag ctgaattttt gcagttaaag catcgtgtag tgtttttctt agtccgttac
     3541 gtaggtagga atctgatgta atggttgttg gtattttgtc accattcatt tttatctggt
     3601 tgttctcaag ttcggttacg agatccattt gtctatctag ttcaacttgg aaaatcaacg
     3661 tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat attgctgtaa gtgtttaaat
     3721 ctttacttat tggtttcaaa acccattggt taagcctttt aaactcatgg tagttatttt
     3781 caagcattaa catgaactta aattcatcaa ggctaatctc tatatttgcc ttgtgagttt
     3841 tcttttgtgt tagttctttt aataaccact cataaatcct catagagtat ttgttttcaa
     3901 aagacttaac atgttccaga ttatatttta tgaatttttt taactggaaa agataaggca
     3961 atatctcttc actaaaaact aattctaatt tttcgcttga gaacttggca tagtttgtcc
     4021 actggaaaat ctcaaagcct ttaaccaaag gattcctgat ttccacagtt ctcgtcatca
     4081 gctctctggt tgctttagct aatacaccat aagcattttc cctactgatg ttcatcatct
     4141 gagcgtattg gttataagtg aacgataccg tccgttcttt ccttgtaggg ttttcaatcg
     4201 tggggttgag tagtgccaca cagcataaaa ttagcttggt ttcatgctcc gttaagtcat
     4261 agcgactaat cgctagttca tttgctttga aaacaactaa ttcagacata catctcaatt
     4321 ggtctaggtg attttaatca ctataccaat tgagatgggc tagtcaatga taattactag
     4381 tccttttcct ttgagttgtg ggtatctgta aattctgcta gacctttgct ggaaaacttg
     4441 taaattctgc tagaccctct gtaaattccg ctagaccttt gtgtgttttt tttgtttata
     4501 ttcaagtggt tataatttat agaataaaga aagaataaaa aaagataaaa agaatagatc
     4561 ccagccctgt gtataactca ctactttagt cagttccgca gtattacaaa aggatgtcgc
     4621 aaacgctgtt tgctcctcta caaaacagac cttaaaaccc taaaggctta agtagcaccc
     4681 tcgcaagctc gggcaaatcg ctgaatattc cttttgtctc cgaccatcag gcacctgagt
     4741 cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg ctctggcagt gaatgggggt
     4801 aaatggcact acaggcgcct tttatggatt catgcaagga aactacccat aatacaagaa
     4861 aagcccgtca cgggcttctc agggcgtttt atggcgggtc tgctatgtgg tgctatctga
     4921 ctttttgctg ttcagcagtt cctgccctct gattttccag tctgaccact tcggattatc
     4981 ccgtgacagg tcattcagac tggctaatgc acccagtaag gcagcggtat catcaacagg
     5041 cttacccgtc ttactgtcaa gaggacatcc ggtcaaataa aacgaaaggc tcagtcgaaa
     5101 gactgggcct ttcgttttgc tgaggagact tagggaccct gctcttctgg ctaatggaga
     5161 ttttcaacat gggctagcac agccctaggt attatgctag cgtggtgtct gcgtaataag
     5221 gagtcttaat catgccagtt cttttgggta ttccgttatt attgcgtttc ctcggtttcc
     5281 ttctggtaac tttgttcggc tatctgctta cttttctcaa aaagggcttc ggtaagatag
     5341 ctattgctat ttcattgttt cttgctctta ttattgggct taactcaatt cttgtgggtt
     5401 atctctctga tattagtgct caattaccct ctgactttgt tcagggtgtt cagttaattc
     5461 tcccgtctaa tgcgcttccc tgtttttatg ttattctctc tgtaaagact gctattttca
     5521 tttttgacgt taaacaaaaa atcgtttctt atttggattg ggacaaataa tatggctgtt
     5581 tattttgtaa ctggcaaatt aggctctgga aagacgctcg ttagcgttgg taagattcag
     5641 gataaaattg tagctgggtg caaaatagca actaatcttg atttaaggct tcaaaacctc
     5701 ccgcaagtcg ggaggttcgc taaaacgcct cgcgttctta gaataccgga taagccttct
     5761 atatctgatt tgcttgctat tgggcgcggt aatgattcct acgatgaaaa taaaaacgga
     5821 ttgcttgttc tcgatgagtg cggtacttgg tttaataccc gttcttggaa tgataaggaa
     5881 agacagccga ttattgattg gtttctacat gctcgtaaat taggatggga tattattttt
     5941 cttgttcagg acttatctat tgttgataaa caggcgcgtt ctgcattagc tgaacatgtt
     6001 gtttattgtc gtcgtctgga cagaattact ttaccttttg tcggtacttt atattctctt
     6061 attactggct cgaaaatgcc tctgcctaaa ttacatgttg gcgttgttaa atatggcgat
     6121 tctcaattaa gccctactgt tgagcgttgg ctttatactg gtaagaattt gtataacgca
     6181 tacgatacta aacaggcttt ttctagtaat tatgattccg gtgtttattc ttatttaacg
     6241 ccttatttat cacacggtcg gtatttcaaa ccattaaatt taggtcagaa gatgaaatta
     6301 actaaaatat atttgaaaaa gttttctcgc gttctttgtc ttgcgattgg atttgcatca
     6361 gcatttacat atagttatat aacccaacct aagccggagg ttaaaaaggt agtctctcag
     6421 acctatgatt ttgataaatt cactattgac tcttctcatc gtcttaatct aagctatcgc
     6481 tatgttttca aggattctaa gggaaaatta attaatagcg acgatttaca gaagcaaggt
     6541 tattcactta catatattga tttatgtact gtttccatta aaaaaggtaa ttcaaatgaa
     6601 attgttaaat gtaattaatt ttgttttctt gatgtttgtt tcatcatctt cttttgctca
     6661 ggtaattgaa atgaataatt cgcctctgcg cgattttata acttggtatt caaagcaatc
     6721 aggcgaatcc gttattgttt ctcccgatgt aaaaggtact gttactgtat attcatctga
     6781 cgttaaacct gaaaatctac gcaatttctt tatttctgtt ttacgtgcaa ataattttga
     6841 tatggtaggt tctaaccctt ccattattca gaagtataat c
//
