LOCUS       sequence-368055-        5197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses retron Eco1 ncRNA v32, and Cas1 + Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5197)
  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 5197)
  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: pSLS.407
FEATURES             Location/Qualifiers
     source          1..5197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             188..205
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        319..337
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    338..362
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             1734..1778
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1816..1834)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1830..1877
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2107..2922)
                     /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(2107..2922)
                     /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     2830..2849
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2923..3014)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3030..3779)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(3040..3057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3280..3299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3975..5057)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAVVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHKQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5019..5038
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5058..5135)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5181..5197,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcatttaa ctatgataag attccgtatg cgcaccctta gcgagaggtt tatcattaag
       61 gtcaacctct ggatgttgtt tcggcatcct gcattgaatc tgagttacaa gctgtttgtc
      121 gccagtcaga ctggcgacaa cccgtttttt ctgacgtaag ggtgcgcaac tttcatgaaa
      181 tccgctgcat caccatcatc accacagcca ggatccgaat tcgagctcgg cgcgcctgca
      241 ggtcgacaag cttgcggccg cataatgctt aagtcgaaca gaaagtaatc gtattgtaca
      301 cggccgcata atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
      361 ccccatctta gtatattagt taagtataag aaggagatat acaatgacct ggcttcccct
      421 taatcccatt ccactcaaag atcgcgtctc catgatcttt ctgcaatatg ggcagatcga
      481 tgtaatagat ggcgcgtttg tacttatcga caagacaggg atccgcactc atattcctgt
      541 tggctcggtt gcctgcatca tgctggaacc tggtacacgg gtttcgcatg cagctgtacg
      601 cctggctgcg caagttggaa cattgttggt atgggtgggg gaagcgggcg ttcgtgttta
      661 tgcttctggt cagcctggag gtgcgcgttc agataagctg ctctatcagg caaaacttgc
      721 tctggatgaa gatttgcgtc tgaaggtcgt acgtaaaatg tttgaacttc ggtttggaga
      781 acctgcgcct gcccggcgct ccgtagagca actcagaggt atagaaggca gtcgcgtgcg
      841 ggcaacctac gcacttctgg cgaagcaata cggcgtgaca tggaatggac gtcgctacga
      901 tccgaaagac tgggaaaagg gcgatacgat caaccaatgc attagcgctg caacttcctg
      961 tttatacggc gtaactgaag cggcgatact tgcagctggt tatgcaccag ctattgggtt
     1021 tgtgcataca ggaaagcctc tttcctttgt ttacgatatt gcagacatca ttaaatttga
     1081 cactgttgta ccgaaagctt ttgagatagc gcgtcgtaac cctggtgagc cggaccggga
     1141 agtccgtttg gcgtgcaggg atatttttcg cagtagtaaa acattagcca aattgattcc
     1201 gcttatagag gacgtgcttg ccgctggaga aatacaaccg ccggccccac ctgaagatgc
     1261 acagcctgtt gccattccgc ttcctgtttc actgggagat gcaggccatc ggagtagctg
     1321 aaatgagtat gttggtcgtg gtcactgaaa atgtacctcc gcgcttacga ggcagattag
     1381 ccatctggtt gttggaggta cgtgcagggg tatatgtagg tgatgtatcc gcaaaaattc
     1441 gtgaaatgat ctgggaacaa atagctggac tggcggaaga aggcaatgta gtgatggcat
     1501 gggcaacgaa tacagaaacg ggatttgagt tccagacatt tgggttaaac aggcgtaccc
     1561 cggtagattt ggatggttta aggttggtgt cttttttacc tgtttgagcg gccgcactcg
     1621 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacattatg gcagatctca
     1681 attggatatc ggccggccac gcgatcgctg acgtcggtac cctcgagtct ggtaaagaaa
     1741 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     1801 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1861 tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc
     1921 cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     1981 ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg
     2041 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     2101 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     2161 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     2221 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     2281 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     2341 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     2401 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2461 tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg
     2521 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     2581 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     2641 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     2701 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2761 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     2821 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     2881 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     2941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3001 gaaaaataaa caaataggca tgcagcgctc ttccgcttcc tcgctcactg actcgctacg
     3061 ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa tacggttatc
     3121 cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag caccggaaga
     3181 agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3241 gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacatgtccg
     3361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg tatctcagtt
     3421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3541 cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt atgcacctgt
     3601 taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca cttaccttgg
     3661 ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc ggtggttttt
     3721 ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc tattccgtta
     3781 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     3841 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3901 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3961 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4021 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4081 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4141 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4201 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     4261 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4321 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4381 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4441 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4501 gctaacagcg cgatttgctt gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4561 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4621 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4681 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccac cgctttacag
     4741 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4801 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4861 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4921 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4981 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5041 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5101 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5161 cgactcctgc attaggaaat taatacgact cactata
//
