LOCUS       sequence-368053-        6192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wild-type retron Eco1 ncRNA + Eco1 RT, and Cas1 + Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6192)
  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 6192)
  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.405
FEATURES             Location/Qualifiers
     source          1..6192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1183..1200
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1314..1332
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1333..1357
                     /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             2729..2773
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2811..2829)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2825..2872
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3102..3917)
                     /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(3102..3917)
                     /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     3825..3844
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3918..4009)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4025..4774)
                     /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(4035..4052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4275..4294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4970..6052)
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6014..6033
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6053..6130)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6176..6192,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcatttaa ctatgataag attccgtatg cgcaccctta gcgagaggtt tatcattaag
       61 gtcaacctct ggatgttgtt tcggcatcct gcattgaatc tgagttactg tctgttttcc
      121 ttgttggaac ggagagcatc gcctgatgct ctccgagcca accaggaaac ccgttttttc
      181 tgacgtaagg gtgcgcaact ttcatgaaat ccgctgaata tttgaacact tttagattga
      241 gaaatctcgg cctacctgtc atgaacaatt tgcatgacat gtctaaggcg actcgcatat
      301 ctgttgaaac acttcggttg ttaatctata cagctgattt tcgctatagg atctacactg
      361 tagaaaagaa aggcccagag aagagaatga gaaccattta ccaaccttct cgagaactta
      421 aagccttaca aggatgggtt ctacgtaaca ttttagataa actgtcgtca tctccttttt
      481 ctattggatt tgaaaagcac caatctattt tgaataatgc taccccgcat attggggcaa
      541 actttatact gaatattgat ttggaggatt ttttcccaag tttaactgct aacaaagttt
      601 ttggagtgtt ccattctctt ggttataatc gactaatatc ttcagttttg acaaaaatat
      661 gttgttataa aaatctgcta ccacaaggtg ctccatcatc acctaaatta gctaatctaa
      721 tatgttctaa acttgattat cgtattcagg gttatgcagg tagtcggggc ttgatatata
      781 cgagatatgc cgatgatctc accttatctg cacagtctat gaaaaaggtt gttaaagcac
      841 gtgatttttt attttctata atcccaagtg aaggattggt tattaactca aaaaaaactt
      901 gtattagtgg gcctcgtagt cagaggaaag ttacaggttt agttatttca caagagaaag
      961 ttgggatagg tagagaaaaa tataaagaaa ttagagcaaa gatacatcat atattttgcg
     1021 gtaagtcttc tgagatagaa cacgttaggg gatggttgtc atttatttta agtgtggatt
     1081 caaaaagcca taggagatta ataacttata ttagcaaatt agaaaaaaaa tatggaaaga
     1141 accctttaaa taaagcgaag acctaatgac atgggcagca gccatcacca tcatcaccac
     1201 agccaggatc cgaattcgag ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa
     1261 tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga aattaatacg
     1321 actcactata ggggaattgt gagcggataa caattcccca tcttagtata ttagttaagt
     1381 ataagaagga gatatacaat gacctggctt ccccttaatc ccattccact caaagatcgc
     1441 gtctccatga tctttctgca atatgggcag atcgatgtaa tagatggcgc gtttgtactt
     1501 atcgacaaga cagggatccg cactcatatt cctgttggct cggttgcctg catcatgctg
     1561 gaacctggta cacgggtttc gcatgcagct gtacgcctgg ctgcgcaagt tggaacattg
     1621 ttggtatggg tgggggaagc gggcgttcgt gtttatgctt ctggtcagcc tggaggtgcg
     1681 cgttcagata agctgctcta tcaggcaaaa cttgctctgg atgaagattt gcgtctgaag
     1741 gtcgtacgta aaatgtttga acttcggttt ggagaacctg cgcctgcccg gcgctccgta
     1801 gagcaactca gaggtataga aggcagtcgc gtgcgggcaa cctacgcact tctggcgaag
     1861 caatacggcg tgacatggaa tggacgtcgc tacgatccga aagactggga aaagggcgat
     1921 acgatcaacc aatgcattag cgctgcaact tcctgtttat acggcgtaac tgaagcggcg
     1981 atacttgcag ctggttatgc accagctatt gggtttgtgc atacaggaaa gcctctttcc
     2041 tttgtttacg atattgcaga catcattaaa tttgacactg ttgtaccgaa agcttttgag
     2101 atagcgcgtc gtaaccctgg tgagccggac cgggaagtcc gtttggcgtg cagggatatt
     2161 tttcgcagta gtaaaacatt agccaaattg attccgctta tagaggacgt gcttgccgct
     2221 ggagaaatac aaccgccggc cccacctgaa gatgcacagc ctgttgccat tccgcttcct
     2281 gtttcactgg gagatgcagg ccatcggagt agctgaaatg agtatgttgg tcgtggtcac
     2341 tgaaaatgta cctccgcgct tacgaggcag attagccatc tggttgttgg aggtacgtgc
     2401 aggggtatat gtaggtgatg tatccgcaaa aattcgtgaa atgatctggg aacaaatagc
     2461 tggactggcg gaagaaggca atgtagtgat ggcatgggca acgaatacag aaacgggatt
     2521 tgagttccag acatttgggt taaacaggcg taccccggta gatttggatg gtttaaggtt
     2581 ggtgtctttt ttacctgttt gagcggccgc actcgagtct ggtaaagaaa ccgctgctgc
     2641 gaaatttgaa cgccagcaca ttatggcaga tctcaattgg atatcggccg gccacgcgat
     2701 cgctgacgtc ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca
     2761 gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca
     2821 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     2881 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     2941 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc
     3001 gggtctccgc aagtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3061 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     3121 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     3181 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     3241 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     3301 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     3361 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     3421 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     3481 acgcggtcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     3541 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     3601 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     3661 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     3721 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3781 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3841 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     3901 cgttgaatat ggctcatact cttccttttt caatattatt gaagcattta tcagggttat
     3961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggcatgcag
     4021 cgctcttccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     4081 gtgtcagctc actcaaaagc ggtaatacgg ttatccacag aatcagggga taaagccgga
     4141 aagaacatgt gagcaaaaag caaagcaccg gaagaagcca acgccgcagg cgtttttcca
     4201 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagccaga ggtggcgaaa
     4261 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4321 tgttccgacc ctgccgctta ccggatacat gtccgccttt ctcccttcgg gaagcgtggc
     4381 gctttctcat agctcacgct gttggtatct cagttcggtg taggtcgttc gctccaagct
     4441 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4501 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ttggtaactg
     4561 atttagagga ctttgtcttg aagttatgca cctgttaagg ctaaactgaa agaacagatt
     4621 ttggtgagtg cggtcctcca acccacttac cttggttcaa agagttggta gctcagcgaa
     4681 ccttgagaaa accaccgttg gtagcggtgg tttttcttta tttatgagat gatgaatcaa
     4741 tcggtctatc aagtcaacga acagctattc cgttactcta gatttcagtg caatttatct
     4801 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     4861 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4921 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4981 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5041 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5101 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5161 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5221 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     5281 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5341 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5401 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5461 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5521 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5581 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5641 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5701 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5761 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5821 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5881 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5941 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6001 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6061 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6121 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata
     6181 cgactcacta ta
//
