LOCUS       sequence-367486-        4204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Eco1 ncRNA v35 (long a1/a2) and Eco1 ncRNA v35 (long 
            a1/a2, barcode 6) from promoters pTet* and pBetI, respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4204)
  AUTHORS   Bhattarai-Kline S, Lear SK, Fishman CB, Lopez SC, Lockshin ER, 
            Schubert MG, Nivala J, Church GM, Shipman SL
  TITLE     Recording gene expression order in DNA by CRISPR addition of retron 
            barcodes.
  JOURNAL   Nature. 2022 Aug;608(7921):217-225. doi: 10.1038/s41586-022-04994-6.
            Epub 2022 Jul 27.
  PUBMED    35896746
REFERENCE   2  (bases 1 to 4204)
  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/s41586-022-04994-6"; journalName: "Nature"; date: 
            "2022-08"; volume: "608"; issue: "7921"; pages: "217-225"
COMMENT     Sequence Label: pSBK.134
FEATURES             Location/Qualifiers
     source          1..4204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    109..127
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    134..152
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             503..1126
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(576..595)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(1316..1335)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     terminator      1506..1535
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      1557..2144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2045..2064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2156..2183)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2207..2398)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2399..3058)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2973..2992
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3059..3149)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(3150..3181)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 cgcgtccgaa cccagctcta agagaatata aaaagccaga ttattaatcc ggctttttta
       61 ttatttcgct taacgatcgt tggctgtttt cagcaggacg cactgacctc cctatcagtg
      121 atagagattg acatccctat cagtgataga gatactgagc acagctgtca ccggatgtgc
      181 tttcctgata agattccgta tgcgcaccct tagcgagagg tttatcatta aggtcaacct
      241 ctggatgttg tttcggcatc ctgcattgaa tctgagttac aagaaaatgg agaggttgca
      301 gcaacctctc cacccgtttt ttctgacgta agggtgcgca tacggaatct tatcactcgg
      361 taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtccaatg
      421 gcggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
      481 gagtcaattc agggtggtga atatgtccag attagataaa agtaaagtga ttaacagcgc
      541 attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa
      601 gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga
      661 cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga
      721 aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca
      781 tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct
      841 cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc
      901 actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt
      961 cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc
     1021 tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat
     1081 catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcctgataag gatcctaatt
     1141 ggtaacgaat cagaattgac ggctcgaggg agtagcatag ggtttgcaga atccctgctt
     1201 cgtccatttg acaggcacat tatgcatcga tgataagctg tcaaacatga gcagatcctc
     1261 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     1321 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcaaatat
     1381 tttatctgag gtgcttcctc gctcactgct gaaaggagga actatatccg ggtaacgaat
     1441 tcaagcttga tatcattcag gacgagcctc agactccagc gtaactggac tgcaatcaac
     1501 tcactggctc accttcacgg gtgggccttt cttcggtaga aaatcaaagg atcttcttga
     1561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1621 gtggtttgtt tgccggatca agagctacca actctttttc cgaggtaact ggcttcagca
     1681 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     1741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1981 aggcggacag gtatccggta agcggcagtg tcggaacagg agagcgcacg agggagcttc
     2041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2101 atcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcag
     2161 aaaggcccac ccgaaggtga gccaggtgat tacatttggg ccctcatcag aggttttcac
     2221 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     2281 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     2341 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcattt
     2401 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     2461 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     2521 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca
     2581 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     2641 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     2701 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     2761 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     2821 cgtctttcat tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa
     2881 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     2941 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     3001 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccatag
     3061 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt
     3121 gaaagttgga acctcttacg tgccgatcaa gtcaaaagcc tccggtcgga ggcttttgac
     3181 tttctgctat ggaggtcagg tatgatttaa atgtcaatcg gtcggcagat gctgggtgat
     3241 aaaatgacgg gtcaggctat ttgcacgggt tttatccagc ggtttaccgc tcagtgctgc
     3301 acgcagccac agaccatcaa tcagtgctgc cagaccataa cctgcctctt gtgcctgttc
     3361 acgaggcagt tcacgacgaa attcgctaac cagattgctc agcagacgac gactgctaac
     3421 ctgctgcaga cgatacagca tcggctgatg catgctgatt gcccaaaatg ccagccatgc
     3481 tttcattgct gcgctgctaa cctgggtttc atcaaaatta ccaccaacaa ttgcctgcag
     3541 acgctgttct gcgctaccct gcggcagtgc atgcagacga ttcagaactg catcacgcag
     3601 ctggctggta atatcacgca tggttgcttc cagtagaccg tttttatcgc ggaaataatg
     3661 gctaataata ccggtgctaa caccggcacg acgtgcaatc tgtgcaatgg ttgcatcatg
     3721 cataccaact tcattaattg cttccagggt tgcatcaatc agctgacgac gacgaatgct
     3781 ctgcataccc agtttcggca tattcacccc cgtgaattga ctctcttccg ggcgctatca
     3841 tgccataccg cgaaaggttt tgcaccattc gatggcgcgc cgccattgga ccaaaacgaa
     3901 aaaaggcccc cctttcggga ggcctctttt ctggaatttg gtaccgagtg ataagattcc
     3961 gtatgcgcac ccttacgtca gaaaaaacgg gtggagaggt cagtctgacc tctccatttt
     4021 cttgtaactc agattcaatg caggatgccg aaacaacatc cagaggttga ccttaatgat
     4081 aaacctctcg ctaagggtgc gcatacggaa tcttatcagg aaagcacatc cggtgacagc
     4141 tgctagcatt atattgaacg tccaatcaat tgtctattgg taacgaatcc ctctcacccg
     4201 cgct
//
