LOCUS       sequence-233895-        5661 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 5661)
  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 5661)
  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: pBT13-splitD
FEATURES             Location/Qualifiers
     source          1..5661
                     /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
                     FGTTDLVLCSEALLLNFANSLPCLYDLLDVNATTISRIDAAFSARAPNENIAKQVIDHL
                     RNVSNGQTKSTRSQNWESTVTWNETSRHRTLVAYLKHVELQHQIQQLSSKPSAKMTSYQ
                     KEQLKVLSNPDLLEFASGLVRFEARIKTRYLKSFGLPLNLFDAIRFASDYNSQGKDLIF
                     DLWSFSFSELFKAFEGDSMNIYDDSAVLDAIQSKHFTITPSGKTSFAKASRYFGFYRRL
                     VNEGYDSVALTMPRNSFWRYVSALVECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRP
                     DWYNEPVLKIA"
     CDS             1267..1530
                     /codon_start=1
                     /gene="V"
                     /product="pV"
                     /label=M13 gene V
                     /note="replication"
                     /translation="MIKVEIKPSQAQFTTRSGVSRQGKPYSLNEQLCYVDLGNEYPVLV
                     KITLDEGQPAYAPGLYTVHLSSFKVGQFGSLMIDRLRLVPAK"
     CDS             1532..1633
                     /codon_start=1
                     /gene="VII"
                     /product="pVII"
                     /label=M13 gene VII
                     /note="minor coat protein"
                     /translation="MEQVADFDTIYQAMIQISVVLCFALGIIAGGQR"
     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="MKKSLVLKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     protein_bind    2002..2020
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2027..2045
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2063..2074
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2081..2758
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2776..2847
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2863..2890
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2989..3804)
                     /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(2989..3804)
                     /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     3712..3731
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3796..3815
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3872..4822)
                     /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      4870..5092
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 cttattatca accggggtac atatgattga catgctagtt ttacgattac cgttcatcga
       61 ttctcttgtt tgctccagac tctcaggcaa tgacctgata gcctttgtag acctctcaaa
      121 aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atgttgatgg
      181 tgatttgact gtctccggcc tttctcaccc ttttgaatct ttacctacac attactcagg
      241 cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc
      301 ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagttttatg
      361 ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga
      421 tgttaacgct actactatta gtagaattga tgccgccttt tcagctcgcg 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 ggagcaatta aaggtactct ctaatcctga
      721 cctgttggag tttgcttccg gtctggttcg ctttgaagct cgaattaaaa cgcgatattt
      781 gaagtctttc gggcttcctc ttaatctttt tgatgcaatc cgctttgctt ctgactataa
      841 tagtcagggt aaagacctga tttttgattt atggtcattc tcgttttctg aactgtttaa
      901 agcatttgag ggggattcaa tgaatattta tgacgattcc gcagtattgg acgctatcca
      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 aatgagcagc tttgttacgt tgatttgggt
     1381 aatgaatatc cggttcttgt caagattact cttgatgaag gtcagccagc ctatgcgcct
     1441 ggtctgtaca ccgttcatct gtcctctttc aaagttggtc agttcggttc ccttatgatt
     1501 gaccgtctgc gcctcgttcc ggctaagtaa catggagcag gtcgcggatt tcgacacaat
     1561 ttatcaggcg atgatacaaa tctccgttgt actttgtttc gcgcttggta taatcgctgg
     1621 gggtcaaaga tgagtgtttt agtgtattct ttcgcctctt tcgttttagg ttggtgcctt
     1681 cgtagtggca ttacgtattt tacccgttta atggaaactt cctcatgaaa aagtctttag
     1741 tcctcaaagc ctctgtagcc gttgctaccc tcgttccgat gctgtctttc gctgctgagg
     1801 gtgacgatcc cgcaaaagcg gcctttaact ccctgcaagc ctcagcgacc gaatatatcg
     1861 gttatgcgtg ggcgatggtt gttgtcattg tcggcgcaac tatcggtatc aagctgttta
     1921 agaaattcac ctcgaaagca agctgataaa ccgatacaat taaaggctcc ttttggagcc
     1981 ttttttttgg agtagaagag ctccctatca gtgatagaga ttgacatccc tatcagtgat
     2041 agagatactg agcacgaatc tgaaagagga gaaaggatct atggcgagta gcgaagacgt
     2101 tatcaaagag ttcatgcgtt tcaaagttcg tatggaaggt tccgttaacg gtcacgagtt
     2161 cgaaatcgaa ggtgaaggtg aaggtcgtcc gtacgaaggt acccagaccg ctaaactgaa
     2221 agttaccaaa ggtggtccgc tgccgttcgc ttgggacatc ctgtccccgc agttccagta
     2281 cggttccaaa gcttacgtta aacacccggc tgacatcccg gactacctga aactgtcctt
     2341 cccggaaggt ttcaaatggg aacgtgttat gaacttcgaa gacggtggtg ttgttaccgt
     2401 tacccaggac tcctccctgc aagacggtga gttcatctac aaagttaaac tgcgtggtac
     2461 caacttcccg tccgacggtc cggttatgca gaaaaaaacc atgggttggg aagcttccac
     2521 cgaacgtatg tacccggaag acggtgctct gaaaggtgaa atcaaaatgc gtctgaaact
     2581 gaaagacggt ggtcactacg acgctgaagt taaaaccacc tacatggcta aaaaaccggt
     2641 tcagctgccg ggtgcttaca aaaccgacat caaactggac atcacctccc acaacgaaga
     2701 ctacaccatc gttgaacagt acgaacgtgc tgaaggtcgt cactccaccg gtgcttaata
     2761 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     2821 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     2881 cctttctgcg tttatacagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2941 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     3001 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     3061 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     3121 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     3181 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     3241 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     3301 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     3361 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     3421 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     3481 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     3541 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     3601 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     3661 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     3721 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     3781 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     3841 ttttattgtt catgaaggcc aaataggccg ttcagatcct tccgtattta gccagtatgt
     3901 tctctagtgt ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatgct
     3961 tactttgcat gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta
     4021 aagcatcgtg tagtgttttt cttagtccgt tacgtaggta ggaatctgat gtaatggttg
     4081 ttggtatttt gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca
     4141 tttgtctatc tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa
     4201 ccaccaattt catattgctg taagtgttta aatctttact tattggtttc aaaacccatt
     4261 ggttaagcct tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat
     4321 caaggctaat ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc
     4381 actcataaat cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt
     4441 ttatgaattt ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta
     4501 atttttcgct tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca
     4561 aaggattcct gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac
     4621 cataagcatt ttccctactg atgttcatca tctgagcgta ttggttataa gtgaacgata
     4681 ccgtccgttc tttccttgta gggttttcaa tcgtggggtt gagtagtgcc acacagcata
     4741 aaattagctt ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt
     4801 tgaaaacaac taattcagac atacatctca attggtctag gtgattttaa tcactatacc
     4861 aattgagatg ggctagtcaa tgataattac tagtcctttt cctttgagtt gtgggtatct
     4921 gtaaattctg ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt
     4981 ccgctagacc tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa
     5041 agaaagaata aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt
     5101 agtcagttcc gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca
     5161 gaccttaaaa ccctaaaggc ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata
     5221 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc
     5281 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg
     5341 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt
     5401 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc
     5461 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa
     5521 tgcacccagt aaggcagcgg tatcatcaac aggcttaccc gtcttactgt caagaggaca
     5581 tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tgctgaggag
     5641 acttagggac cctgctcttc t
//
