LOCUS       sequence-367487-        5330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wild-type retron Eco4 ncRNA, and Cas1 + Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5330)
  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 5330)
  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.419
FEATURES             Location/Qualifiers
     source          1..5330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             321..338
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        452..470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    471..495
                     /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             1867..1911
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1949..1967)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1963..2010
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2240..3055)
                     /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(2240..3055)
                     /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     2963..2982
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3056..3147)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3163..3912)
                     /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(3173..3190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3413..3432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4108..5190)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5152..5171
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5191..5268)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5314..5330,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccca tttaactatg ataagattcc gtgcgtaaga
       61 tgacaatgct ctttagcgtt ttatggattt accacctgat tggtcaaatc tagttgggcg
      121 ttgcgccaaa ctctaattta ttgattacat ttacagttgc ggaacaaact ttttgagccg
      181 caatccagta ggttgcggat caaaaagttt gttccgcggc ttcaagtaaa gagcctggac
      241 ctagggatgg acagggaact taaagctcct tgttcatctc ttggtactag ttattgactg
      301 gtaaacggaa caagctggta catcaccatc atcaccacag ccaggatccg aattcgagct
      361 cggcgcgcct gcaggtcgac aagcttgcgg ccgcataatg cttaagtcga acagaaagta
      421 atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga
      481 gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga tatacaatga
      541 cctggcttcc ccttaatccc attccactca aagatcgcgt ctccatgatc tttctgcaat
      601 atgggcagat cgatgtaata gatggcgcgt ttgtacttat cgacaagaca gggatccgca
      661 ctcatattcc tgttggctcg gttgcctgca tcatgctgga acctggtaca cgggtttcgc
      721 atgcagctgt acgcctggct gcgcaagttg gaacattgtt ggtatgggtg ggggaagcgg
      781 gcgttcgtgt ttatgcttct ggtcagcctg gaggtgcgcg ttcagataag ctgctctatc
      841 aggcaaaact tgctctggat gaagatttgc gtctgaaggt cgtacgtaaa atgtttgaac
      901 ttcggtttgg agaacctgcg cctgcccggc gctccgtaga gcaactcaga ggtatagaag
      961 gcagtcgcgt gcgggcaacc tacgcacttc tggcgaagca atacggcgtg acatggaatg
     1021 gacgtcgcta cgatccgaaa gactgggaaa agggcgatac gatcaaccaa tgcattagcg
     1081 ctgcaacttc ctgtttatac ggcgtaactg aagcggcgat acttgcagct ggttatgcac
     1141 cagctattgg gtttgtgcat acaggaaagc ctctttcctt tgtttacgat attgcagaca
     1201 tcattaaatt tgacactgtt gtaccgaaag cttttgagat agcgcgtcgt aaccctggtg
     1261 agccggaccg ggaagtccgt ttggcgtgca gggatatttt tcgcagtagt aaaacattag
     1321 ccaaattgat tccgcttata gaggacgtgc ttgccgctgg agaaatacaa ccgccggccc
     1381 cacctgaaga tgcacagcct gttgccattc cgcttcctgt ttcactggga gatgcaggcc
     1441 atcggagtag ctgaaatgag tatgttggtc gtggtcactg aaaatgtacc tccgcgctta
     1501 cgaggcagat tagccatctg gttgttggag gtacgtgcag gggtatatgt aggtgatgta
     1561 tccgcaaaaa ttcgtgaaat gatctgggaa caaatagctg gactggcgga agaaggcaat
     1621 gtagtgatgg catgggcaac gaatacagaa acgggatttg agttccagac atttgggtta
     1681 aacaggcgta ccccggtaga tttggatggt ttaaggttgg tgtctttttt acctgtttga
     1741 gcggccgcac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatt
     1801 atggcagatc tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag
     1861 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
     1921 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     1981 gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg
     2041 gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt
     2101 taacgaccct gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt
     2161 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2221 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2281 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2341 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2401 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2461 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2521 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2581 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac
     2641 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2701 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2761 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2821 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2881 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2941 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3001 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct
     3061 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3121 ttgaatgtat ttagaaaaat aaacaaatag gcatgcagcg ctcttccgct tcctcgctca
     3181 ctgactcgct acgctcggtc gttcgactgc ggcgagcggt gtcagctcac tcaaaagcgg
     3241 taatacggtt atccacagaa tcaggggata aagccggaaa gaacatgtga gcaaaaagca
     3301 aagcaccgga agaagccaac gccgcaggcg tttttccata ggctccgccc ccctgacgag
     3361 catcacaaaa atcgacgctc aagccagagg tggcgaaacc cgacaggact ataaagatac
     3421 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3481 ggatacatgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3541 tggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3601 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3661 cacgacttat cgccactggc agcagccatt ggtaactgat ttagaggact ttgtcttgaa
     3721 gttatgcacc tgttaaggct aaactgaaag aacagatttt ggtgagtgcg gtcctccaac
     3781 ccacttacct tggttcaaag agttggtagc tcagcgaacc ttgagaaaac caccgttggt
     3841 agcggtggtt tttctttatt tatgagatga tgaatcaatc ggtctatcaa gtcaacgaac
     3901 agctattccg ttactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     3961 cagccccata cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag
     4021 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4081 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4141 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4201 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4261 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4321 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4381 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4441 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4501 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4561 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4621 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4681 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     4741 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4801 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     4861 caccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     4921 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     4981 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5041 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5101 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5161 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5221 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5281 gctctccctt atgcgactcc tgcattagga aattaatacg actcactata
//
