LOCUS       sequence-233897-        5662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  clone M13 phage by SapI Golden Gate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5662)
  AUTHORS   Thuronyi BW, Koblan LW, Levy JM, Yeh WH, Zheng C, Newby GA, Wilson 
            C, Bhaumik M, Shubina-Oleinik O, Holt JR, Liu DR
  TITLE     Continuous evolution of base editors with expanded target 
            compatibility and improved activity.
  JOURNAL   Nat Biotechnol. 2019 Jul 22. pii: 10.1038/s41587-019-0193-0. doi: 
            10.1038/s41587-019-0193-0.
  PUBMED    31332326
REFERENCE   2  (bases 1 to 5662)
  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. 2019 Jul 22. pii: 10.1038/s41587-019-0193-0. doi: 
            10.1038/s41587-019-0193-0."
COMMENT     Sequence Label: pBT29-splitD
FEATURES             Location/Qualifiers
     source          1..5662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             23..1255
                     /codon_start=1
                     /gene="II"
                     /product="pII"
                     /label=M13 gene II
                     /note="replication"
                     /translation="MIDMLVLRLPFIDSLVCSRLSGNDLIAFVDLSKIATLSGMNLSAR
                     TVEYHVDGDLTVSGLSHPFESLPTHYSGIAFKIYEGSKNFYPCVEIKASPAKVLQGHNV
                     FGTTDLGLCSEALLLNFANSLPCLYDLLDVNATTISRIDAAFSARAPNENIAKQVIDHL
                     RNVSNGQTKSTRSQNWESTVTWNETSRHRTLVAYLKHVELQHQIQQLSSKPSAKMTSYQ
                     KEQLKVLSNPDLLEFASGLVRFEARIKTRYLKSFGLPLNLFDAIRFASDYNSQGKDLIF
                     DLWSFSFSELFKAFEGNSMNIYDDSAVLAAIQSKHFTITPSGKTSFAKASRYFGFYRRL
                     VNEGYDSVALTMPRNSFWRYVSALVECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRP
                     DWYNEPVLKIA"
     CDS             1267..1530
                     /codon_start=1
                     /gene="V"
                     /product="pV"
                     /label=M13 gene V
                     /note="replication"
                     /translation="MIKVEIKPSQAQFTTRSGVSRQGKPYSLNEQFCYVDLGNEYPVLV
                     KITLDEGQPAYAPGLYTVHLSSFKVGQFGSLMIDRLRLVPAK"
     CDS             1532..1633
                     /codon_start=1
                     /gene="VII"
                     /product="pVII"
                     /label=M13 gene VII
                     /note="minor coat protein"
                     /translation="MEQVADFDIIYQAMIQISVVLCFALGIIAGGQR"
     CDS             1630..1728
                     /codon_start=1
                     /gene="IX"
                     /product="pIX"
                     /label=M13 gene IX
                     /note="minor coat protein"
                     /translation="MSVLVYSFASFVLGWCLRSGITYFTRLMETSS"
     CDS             1725..1946
                     /codon_start=1
                     /gene="VIII"
                     /product="pVIII"
                     /label=M13 gene VIII
                     /note="major coat protein"
                     /translation="MKKSLVLKASVAVATLVPMLSFAAEGDDPAKAAFDSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     terminator      1953..1992
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     protein_bind    2003..2021
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2028..2046
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2064..2075
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2082..2759
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2777..2848
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2864..2891
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2990..3805)
                     /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(2990..3805)
                     /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     3713..3732
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3797..3816
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3873..4823)
                     /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      4871..5093
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 cttattatca accggggtac atatgattga catgctagtt ttacggttac cgttcatcga
       61 ttctcttgtt tgctccagac tctcaggcaa tgacctgata gcctttgtag atctctcaaa
      121 aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atgttgatgg
      181 tgatttgact gtctccggcc tttcccaccc ttttgaatct ttacctacac attactcagg
      241 cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc
      301 ttctcccgca aaagtattac aggggcataa tgtttttggt acaaccgatt taggtttatg
      361 ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga
      421 tgttaacgct actactatta gtagaattga tgccgccttt tcagcccgcg ccccaaatga
      481 aaatatagct aaacaggtta ttgaccattt gcgaaatgta tctaatggtc aaactaaatc
      541 tactcgttcg cagaattggg aatcaactgt tacatggaat gaaacttcca gacaccgtac
      601 tttagttgca tatttaaaac atgttgagct acagcaccag attcagcaat taagctctaa
      661 gccatccgca aaaatgacct cttatcaaaa ggaacaatta aaggtactct ctaatcctga
      721 cctgttggag tttgcttccg gtctggttcg ctttgaggct cgaattaaaa cgagatattt
      781 gaagtctttc gggcttcctc ttaatctttt tgatgcaatc cgctttgctt ctgactataa
      841 tagtcagggt aaagacctga tttttgattt atggtcattc tcgttttctg aactgtttaa
      901 agcatttgag gggaattcaa tgaatattta tgacgattcc gcagtattgg ccgctatcca
      961 gtctaaacat tttactatta ccccctctgg caaaacttct tttgcaaaag cctctcgcta
     1021 ttttggtttt tatcgtcgtc tggtaaacga gggttatgat agtgttgctc ttactatgcc
     1081 tcgtaattcc ttttggcgtt atgtatctgc attagttgaa tgtggtattc ctaaatctca
     1141 actgatgaat ctttctacct gtaataatgt tgttccgtta gttcgtttta ttaacgtaga
     1201 tttttcttcc caacgtcctg actggtataa tgagccagtt cttaaaatcg cataaggtaa
     1261 ttcacaatga ttaaagttga aattaaacca tctcaagccc aatttactac tcgttctggt
     1321 gtttctcgtc agggcaagcc ttattcactg aatgagcagt tttgttacgt tgatttgggt
     1381 aatgaatatc cggttcttgt caagattact cttgatgaag gtcagccagc ctatgcgccg
     1441 ggtctgtaca ccgttcatct gtcctctttc aaagttggtc agttcggttc ccttatgatt
     1501 gaccgtctgc gcctcgttcc ggctaagtaa catggagcag gtcgcggatt tcgacataat
     1561 ttatcaggcg atgatacaaa tctccgttgt actctgtttc gcgcttggta taatagctgg
     1621 gggtcaaaga tgagtgtttt agtgtattct ttcgcctctt tcgttttagg ttggtgcctc
     1681 cgtagtggca ttacgtattt tacccgttta atggaaactt cctcatgaaa aagtctttag
     1741 tcctcaaagc ctctgtagcc gttgctaccc tcgttccgat gctgtctttc gctgctgagg
     1801 gtgacgatcc cgcaaaagcg gcctttgact ccctgcaagc ctcagcgacc gaatatatcg
     1861 gttatgcgtg ggcgatggtt gttgtcattg tcggcgcaac tatcggtatc aagctgttta
     1921 agaaattcac ctcgaaagca agttgataaa ctgatacaat taaaggctcc ttttggagcc
     1981 tttttttttg gagtagaaga gctccctatc agtgatagag attgacatcc ctatcagtga
     2041 tagagatact gagcacgaat ctgaaagagg agaaaggatc tatggcgagt agcgaagacg
     2101 ttatcaaaga gttcatgcgt ttcaaagttc gtatggaagg ttccgttaac ggtcacgagt
     2161 tcgaaatcga aggtgaaggt gaaggtcgtc cgtacgaagg tacccagacc gctaaactga
     2221 aagttaccaa aggtggtccg ctgccgttcg cttgggacat cctgtccccg cagttccagt
     2281 acggttccaa agcttacgtt aaacacccgg ctgacatccc ggactacctg aaactgtcct
     2341 tcccggaagg tttcaaatgg gaacgtgtta tgaacttcga agacggtggt gttgttaccg
     2401 ttacccagga ctcctccctg caagacggtg agttcatcta caaagttaaa ctgcgtggta
     2461 ccaacttccc gtccgacggt ccggttatgc agaaaaaaac catgggttgg gaagcttcca
     2521 ccgaacgtat gtacccggaa gacggtgctc tgaaaggtga aatcaaaatg cgtctgaaac
     2581 tgaaagacgg tggtcactac gacgctgaag ttaaaaccac ctacatggct aaaaaaccgg
     2641 ttcagctgcc gggtgcttac aaaaccgaca tcaaactgga catcacctcc cacaacgaag
     2701 actacaccat cgttgaacag tacgaacgtg ctgaaggtcg tcactccacc ggtgcttaat
     2761 aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     2821 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     2881 gcctttctgc gtttatacag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2941 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     3001 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3061 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3121 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3181 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3241 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3301 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3361 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3421 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3481 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3541 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3601 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3661 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3721 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3781 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3841 gttttattgt tcatgaaggc caaataggcc gttcagatcc ttccgtattt agccagtatg
     3901 ttctctagtg tggttcgttg tttttgcgtg agccatgaga acgaaccatt gagatcatgc
     3961 ttactttgca tgtcactcaa aaattttgcc tcaaaactgg tgagctgaat ttttgcagtt
     4021 aaagcatcgt gtagtgtttt tcttagtccg ttacgtaggt aggaatctga tgtaatggtt
     4081 gttggtattt tgtcaccatt catttttatc tggttgttct caagttcggt tacgagatcc
     4141 atttgtctat ctagttcaac ttggaaaatc aacgtatcag tcgggcggcc tcgcttatca
     4201 accaccaatt tcatattgct gtaagtgttt aaatctttac ttattggttt caaaacccat
     4261 tggttaagcc ttttaaactc atggtagtta ttttcaagca ttaacatgaa cttaaattca
     4321 tcaaggctaa tctctatatt tgccttgtga gttttctttt gtgttagttc ttttaataac
     4381 cactcataaa tcctcataga gtatttgttt tcaaaagact taacatgttc cagattatat
     4441 tttatgaatt tttttaactg gaaaagataa ggcaatatct cttcactaaa aactaattct
     4501 aatttttcgc ttgagaactt ggcatagttt gtccactgga aaatctcaaa gcctttaacc
     4561 aaaggattcc tgatttccac agttctcgtc atcagctctc tggttgcttt agctaataca
     4621 ccataagcat tttccctact gatgttcatc atctgagcgt attggttata agtgaacgat
     4681 accgtccgtt ctttccttgt agggttttca atcgtggggt tgagtagtgc cacacagcat
     4741 aaaattagct tggtttcatg ctccgttaag tcatagcgac taatcgctag ttcatttgct
     4801 ttgaaaacaa ctaattcaga catacatctc aattggtcta ggtgatttta atcactatac
     4861 caattgagat gggctagtca atgataatta ctagtccttt tcctttgagt tgtgggtatc
     4921 tgtaaattct gctagacctt tgctggaaaa cttgtaaatt ctgctagacc ctctgtaaat
     4981 tccgctagac ctttgtgtgt tttttttgtt tatattcaag tggttataat ttatagaata
     5041 aagaaagaat aaaaaaagat aaaaagaata gatcccagcc ctgtgtataa ctcactactt
     5101 tagtcagttc cgcagtatta caaaaggatg tcgcaaacgc tgtttgctcc tctacaaaac
     5161 agaccttaaa accctaaagg cttaagtagc accctcgcaa gctcgggcaa atcgctgaat
     5221 attccttttg tctccgacca tcaggcacct gagtcgctgt ctttttcgtg acattcagtt
     5281 cgctgcgctc acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg
     5341 gattcatgca aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg
     5401 ttttatggcg ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc
     5461 ctctgatttt ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta
     5521 atgcacccag taaggcagcg gtatcatcaa caggcttacc cgtcttactg tcaagaggac
     5581 atccggtcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttgctgagga
     5641 gacttaggga ccctgctctt ct
//
