LOCUS       sequence-254692-        7006 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 7006)
  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 7006)
  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-splitC
FEATURES             Location/Qualifiers
     source          1..7006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    12..30
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    37..55
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             73..84
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             91..768
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      786..857
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      873..900
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(999..1814)
                     /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(999..1814)
                     /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     1722..1741
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1806..1825
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(1882..2832)
                     /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      2880..3102
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3880..4218
                     /codon_start=1
                     /gene="VI"
                     /product="pVI"
                     /label=M13 gene VI
                     /note="minor coat protein"
                     /translation="MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLA
                     LIIGLNSILVGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKTAIFIFDVKQKI
                     VSYLDWDK"
     CDS             4220..5266
                     /codon_start=1
                     /gene="I"
                     /product="pI"
                     /label=M13 gene I
                     /note="phage assembly"
                     /translation="MAVYFVTGKLGSGKTLVSVGKIQDKIVAGCKIATNLDLRLQNLPQ
                     VGRFAKTPRVLRIPDKPSISDLLAIGRGNDSYDENKNGLLVLDECGTWFNTRSWNDKER
                     QPIIDWFLHARKLGWDIIFLVQDLSIVDKQARSALAEHVVYCRRLDRITLPFVGTLYSL
                     ITGSKMPLPKLHVGVVKYGDSQLSPTVERWLYTGKNLYNAYDTKQAFSSNYDSGVYSYL
                     TPYLSHGRYFKPLNLGQKMKLTKIYLKKFSRVLCLAIGFASAFTYSYITQPKPEVKKVV
                     SQTYDFDKFTIDSSHRLNLSYRYVFKDSKGKLINSDDLQKQGYSLTYIDLCTVSIKKGN
                     SNEIVKCN"
     CDS             5244..6524
                     /codon_start=1
                     /gene="IV"
                     /product="pIV"
                     /label=M13 gene IV
                     /note="phage assembly"
                     /translation="MKLLNVINFVFLMFVSSSSFAQVIEMNNSPLRDFITWYSKQSGES
                     VIVSPDVKGTVTVYSSDVKPENLRNFFISVLRANNFDMVGSNPSIIQKYNPNNQDYIDE
                     LPSPDNQEYDDNSAPSGGFFVPQNDNVTQTFKINNVRAKDLIRVVELFVKSNTSKSSNV
                     LSIDGSNLLVVSAPKDILDNLSQFLSTVDLPTDQVLIEGLIFEVQQGDALDFSFAAGSQ
                     RGTVAGGVNTDRLTSVLSSAGGSFGIFNGDVLGLSVRALKTNSHSKILSVPRILTLSGQ
                     KGSISIGQNVPFITGRVTGESANVNNPFQTIERQNVGISMSVFPVAMAGGNIVLDITSK
                     ADSLSSSTQASDVITNQRSIATTVNLRDGQTLLLGGLTDYKNTSQDSGVPFLSKIPLIG
                     LLFSSRSDSNEESTLYVLVKATIVRAL"
     rep_origin      6512..6966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6599..6618)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6808..6829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cttagaagag ctccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg
       61 agcacgaatc tgaaagagga gaaaggatct atggcgagta gcgaagacgt tatcaaagag
      121 ttcatgcgtt tcaaagttcg tatggaaggt tccgttaacg gtcacgagtt cgaaatcgaa
      181 ggtgaaggtg aaggtcgtcc gtacgaaggt acccagaccg ctaaactgaa agttaccaaa
      241 ggtggtccgc tgccgttcgc ttgggacatc ctgtccccgc agttccagta cggttccaaa
      301 gcttacgtta aacacccggc tgacatcccg gactacctga aactgtcctt cccggaaggt
      361 ttcaaatggg aacgtgttat gaacttcgaa gacggtggtg ttgttaccgt tacccaggac
      421 tcctccctgc aagacggtga gttcatctac aaagttaaac tgcgtggtac caacttcccg
      481 tccgacggtc cggttatgca gaaaaaaacc atgggttggg aagcttccac cgaacgtatg
      541 tacccggaag acggtgctct gaaaggtgaa atcaaaatgc gtctgaaact gaaagacggt
      601 ggtcactacg acgctgaagt taaaaccacc tacatggcta aaaaaccggt tcagctgccg
      661 ggtgcttaca aaaccgacat caaactggac atcacctccc acaacgaaga ctacaccatc
      721 gttgaacagt acgaacgtgc tgaaggtcgt cactccaccg gtgcttaata aggatctcca
      781 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
      841 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
      901 tttatacagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
      961 aaatacattc aaatatgtat ccgctcataa attaattctt agaaaaactc atcgagcatc
     1021 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1081 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1141 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1201 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1261 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1321 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1381 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1441 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1501 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1561 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1621 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1681 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1741 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     1801 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     1861 catgaaggcc aaataggccg ttcagatcct tccgtattta gccagtatgt tctctagtgt
     1921 ggttcgttgt ttttgcgtga gccatgagaa cgaaccattg agatcatgct tactttgcat
     1981 gtcactcaaa aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg
     2041 tagtgttttt cttagtccgt tacgtaggta ggaatctgat gtaatggttg ttggtatttt
     2101 gtcaccattc atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc
     2161 tagttcaact tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt
     2221 catattgctg taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct
     2281 tttaaactca tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat
     2341 ctctatattt gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat
     2401 cctcatagag tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt
     2461 ttttaactgg aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct
     2521 tgagaacttg gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct
     2581 gatttccaca gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt
     2641 ttccctactg atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc
     2701 tttccttgta gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt
     2761 ggtttcatgc tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac
     2821 taattcagac atacatctca attggtctag gtgattttaa tcactatacc aattgagatg
     2881 ggctagtcaa tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg
     2941 ctagaccttt gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc
     3001 tttgtgtgtt ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata
     3061 aaaaaagata aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc
     3121 gcagtattac aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa
     3181 ccctaaaggc ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt
     3241 ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca
     3301 cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa
     3361 ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg
     3421 gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc
     3481 cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt
     3541 aaggcagcgg tatcatcaac aggcttaccc gtcttactgt caagaggaca tccggtcaaa
     3601 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tgctgaggag acttagggac
     3661 cctgctcttc tggctctaga aggagatttt caacatgctc cctcaatcgg ttgaatgtcg
     3721 cccttttgtc tttggcgctg gtaaaccata tgaattttct attgattgtg acaaaataaa
     3781 cttattccgt ggtgtctttg cgtttctttt atatgttgcc acctttatgt atgtatcttc
     3841 tacgtttgct aacatactgc gtaataagga gtcttaatca tgccagttct tttgggtatt
     3901 ccgttattat tgcgtttcct cggtttcctt ctggtaactt tgttcggcta tctgcttact
     3961 tttctcaaaa agggcttcgg taagatagct attgctattt cattgtttct tgctcttatt
     4021 attgggctta actcaattct tgtgggttat ctctctgata ttagtgctca attaccctct
     4081 gactttgttc agggtgttca gttaattctc ccgtctaatg cgcttccctg tttttatgtt
     4141 attctctctg taaagactgc tattttcatt tttgacgtta aacaaaaaat cgtttcttat
     4201 ttggattggg acaaataata tggctgttta ttttgtaact ggcaaattag gctctggaaa
     4261 gacgctcgtt agcgttggta agattcagga taaaattgta gctgggtgca aaatagcaac
     4321 taatcttgat ttaaggcttc aaaacctccc gcaagtcggg aggttcgcta aaacgcctcg
     4381 cgttcttaga ataccggata agccttctat atctgatttg cttgctattg ggcgcggtaa
     4441 tgattcctac gatgaaaata aaaacggatt gcttgttctc gatgagtgcg gtacttggtt
     4501 taatacccgt tcttggaatg ataaggaaag acagccgatt attgattggt ttctacatgc
     4561 tcgtaaatta ggatgggata ttatttttct tgttcaggac ttatctattg ttgataaaca
     4621 ggcgcgttct gcattagctg aacatgttgt ttattgtcgt cgtctggaca gaattacttt
     4681 accttttgtc ggtactttat attctcttat tactggctcg aaaatgcctc tgcctaaatt
     4741 acatgttggc gttgttaaat atggcgattc tcaattaagc cctactgttg agcgttggct
     4801 ttatactggt aagaatttgt ataacgcata cgatactaaa caggcttttt ctagtaatta
     4861 tgattccggt gtttattctt atttaacgcc ttatttatca cacggtcggt atttcaaacc
     4921 attaaattta ggtcagaaga tgaaattaac taaaatatat ttgaaaaagt tttctcgcgt
     4981 tctttgtctt gcgattggat ttgcatcagc atttacatat agttatataa cccaacctaa
     5041 gccggaggtt aaaaaggtag tctctcagac ctatgatttt gataaattca ctattgactc
     5101 ttctcatcgt cttaatctaa gctatcgcta tgttttcaag gattctaagg gaaaattaat
     5161 taatagcgac gatttacaga agcaaggtta ttcacttaca tatattgatt tatgtactgt
     5221 ttccattaaa aaaggtaatt caaatgaaat tgttaaatgt aattaatttt gttttcttga
     5281 tgtttgtttc atcatcttct tttgctcagg taattgaaat gaataattcg cctctgcgcg
     5341 attttataac ttggtattca aagcaatcag gcgaatccgt tattgtttct cccgatgtaa
     5401 aaggtactgt tactgtatat tcatctgacg ttaaacctga aaatctacgc aatttcttta
     5461 tttctgtttt acgtgcaaat aattttgata tggtaggttc taacccttcc attattcaga
     5521 agtataatcc aaacaatcag gattatattg atgaattgcc atcacctgat aatcaggaat
     5581 atgatgataa ttccgctcct tctggtggtt tctttgtccc gcaaaatgat aatgttactc
     5641 aaacttttaa aattaataac gttcgggcaa aggatttaat acgagttgtc gaattgtttg
     5701 taaagtctaa tacttctaaa tcctcaaatg tattatctat tgacggatct aatctattag
     5761 ttgttagtgc tcctaaagat attttagata acctttctca attcctttca actgttgatt
     5821 tgccaactga ccaggtattg attgagggtt tgatatttga ggttcagcaa ggtgatgctt
     5881 tagatttttc atttgctgct ggctctcagc gtggcactgt tgcaggcgga gttaatactg
     5941 accgcctcac ctctgtttta tcttctgctg gtggttcgtt cggtattttt aatggcgatg
     6001 ttttagggct atcagttcgc gcattaaaga ctaatagcca ttcaaaaata ttgtctgtgc
     6061 cacgtattct tacgctttca ggtcagaagg gttctatctc tattggccag aatgtccctt
     6121 ttattactgg tcgtgtaact ggtgaatctg ccaatgtaaa taatccattt cagacgattg
     6181 agcgtcaaaa tgtaggtatt tccatgagcg tttttcctgt tgcaatggct ggcggtaata
     6241 ttgttctgga tattaccagc aaggccgata gtttgagttc ttctactcag gcaagtgatg
     6301 ttattactaa ccaaagaagt attgctacaa cggttaattt gcgtgatgga cagactcttc
     6361 tactcggtgg cctcactgat tataaaaaca cttctcagga ttctggcgta ccgttcctgt
     6421 ctaaaatccc tttaatcggc ctcctgttta gctcccgctc tgattctaac gaggagagca
     6481 cgttatacgt gctcgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     6541 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     6601 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagcgcta
     6661 aatcgggggc ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     6721 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     6781 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     6841 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6901 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     6961 caatttaaat atttgcttat acaatcttcc tgtttttggg gctttt
//
