LOCUS       sequence-368082-        4574 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 pSalTTC and pTet*, respectively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4574)
  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 4574)
  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.136
FEATURES             Location/Qualifiers
     source          1..4574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1637..1656)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     terminator      1827..1856
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      1878..2465
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2366..2385
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2477..2504)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2528..2719)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2720..3379)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3294..3313
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3380..3470)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(3471..3502)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(3535..4158)
                     /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     4066..4085
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    4509..4527
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4534..4552
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 cgcgtccgaa cccagctcta agagaatata aaaagccaga ttattaatcc ggctttttta
       61 ttatttcgct taacgatcgt tggctggggg cctcgcttgg gttattgctg gtgcccggcc
      121 gggcgcaata ttaatgttga tgatttatta tatatcgagt ggtgtattta tttatattgt
      181 ttgctccgtt accgttatta acagctgtca ccggatgtgc tttcctgata agattccgta
      241 tgcgcaccct tagcgagagg tttatcatta aggtcaacct ctggatgttg tttcggcatc
      301 ctgcattgaa tctgagttac aagaaaatgg agaggttgca gcaacctctc cacccgtttt
      361 ttctgacgta agggtgcaca tacggaatct tatcactcgg taccaaattc cagaaaagag
      421 gcctcccgaa aggggggcct ttttcgtttt ggtccaatgg cggcgcgcca tcgaatggtg
      481 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca tgggggttga
      541 atatggaact gcgtgacctg gatttaaacc tgctggtggt gttcaaccag ttgctggtcg
      601 acagacgcgt ctctgtcact gcggagaacc tgggcctgac ccagcctgcc gtgagcaatg
      661 cgctgaaacg cctgcgcacc tcgctacagg acccactctt cgtgcgcaca catcagggaa
      721 tggaacccac accctatgcc gcgcatctgg ccgagcacgt cacttcggcc atgcacgcac
      781 tgcgcaacgc cctacagcac catgaaagct tcgatccgct gaccagcgag cgtaccttca
      841 ccctggccat gaccgacatt ggcgagatct acttcatgcc gcggctgatg gatgcgctgg
      901 ctcaccaggc ccccaattgc gtgatcagta cggtgcgcga cagttcgatg agcctgatgc
      961 aggccttgca gaacggaacc gtggacttgg ccgtgggcct gcttcccaat ctgcaaactg
     1021 gcttctttca gcgccggctg ctccagaatc actacgtgtg cctatgtcgc aaggaccatc
     1081 cagtcacccg cgaacccctg actctggagc gcttctgttc ctacggccac gtgcgtgtca
     1141 tcgccgctgg caccggccac ggcgaggtgg acacgtacat gacacgggtc ggcatccggc
     1201 gcgacatccg tctggaagtg ccgcacttcg ccgccgttgg ccacatcctc cagcgcaccg
     1261 atctgctcgc cactgtgccg atatgtttag ccgactgctg cgtagagccc ttcggcctaa
     1321 gcgccttgcc gcacccagtc gtcttgcctg aaatagccat caacatgttc tggcatgcga
     1381 agtaccacaa ggacctagcc aatatttggt tgcggcaact gatgtttgac ctgtttacgg
     1441 attgataagg atcctaattg gtaacgaatc agacaattga cggctcgagg gagtagcata
     1501 gggtttgcag aatccctgct tcgtccattt gacaggcaca ttatgcatcg atgataagct
     1561 gtcaaacatg agcagatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     1621 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     1681 ttcgggctca tgagcaaata ttttatctga ggtgcttcct cgctcactgc tgaaaggagg
     1741 aactatatcc gggtaacgaa ttcaagcttg atatcattca ggacgagcct cagactccag
     1801 cgtaactgga ctgcaatcaa ctcactggct caccttcacg ggtgggcctt tcttcggtag
     1861 aaaatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1921 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1981 ccgaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2041 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2101 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2161 gatagttacc ggataaggcg cagtggtcgg gctgaacggg gggttcgtgc acacagccca
     2221 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2281 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2341 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2401 ttcgccacct ctgacttgag catcgatttt tgtgatgctc gtcagggggg cggagcctat
     2461 ggaaaaacgc cagcaacgca gaaaggccca cccgaaggtg agccaggtga ttacatttgg
     2521 gccctcatca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2581 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2641 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2701 ttttttcctg tttggtcatt tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     2761 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     2821 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga
     2881 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     2941 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     3001 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     3061 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     3121 tatcccatat caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca
     3181 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     3241 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     3301 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     3361 cagtgatttt tttctccata gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     3421 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc
     3481 ctccggtcgg aggcttttga ctttctgcta tggaggtcag gtatgattta aatgtcagga
     3541 cccactttca catttaagtt gtttttctaa tccgcatatg atcaattcaa ggccgaataa
     3601 gaaggctggc tctgcacctt ggtgatcaaa taattcgata gcttgtcgta ataatggcgg
     3661 catactatca gtagtaggtg tttccctttc ttctttagcg acttgatgct cttgatcttc
     3721 caatacgcaa cctaaagtaa aatgccccac agcgctgagt gcatataatg cattctctag
     3781 tgaaaaacct tgttggcata aaaaggctaa ttgattttcg agagtttcat actgtttttc
     3841 tgtaggccgt gtacctaaat gtacttttgc tccatcgcga tgacttagta aagcacatct
     3901 aaaactttta gcgttattac gtaaaaaatc ttgccagctt tccccttcta aagggcaaaa
     3961 gtgagtatgg tgcctatcta acatctcaat ggctaaggcg tcgagcaaag cccgcttatt
     4021 ttttacatgc caatacaatg taggctgctc tacacctagc ttctgggcga gtttacgggt
     4081 tgttaaacct tcgattccga cctcattaag cagctctaat gcgctgttaa tcactttact
     4141 tttatctaat ctggacatat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4201 cataccgcga aaggttttgc gccattcgat ggcgcgccgc cattggacca aaacgaaaaa
     4261 aggcccccct ttcgggaggc ctcttttctg gaatttggta ccgagtgata agattccgta
     4321 tgcgcaccct tacgtcagaa aaaacgggtg gagaggtcag tctgacctct ccattttctt
     4381 gtaactcaga ttcaatgcag gatgccgaaa caacatccag aggttgacct taatgataaa
     4441 cctctcgcta agggtgcgca tacggaatct tatcaggaaa gcacatccgg tgacagctgt
     4501 gctcagtatc tctatcactg atagggatgt caatctctat cactgatagg gaggtcagtg
     4561 cgtcctgctg aaaa
//
