LOCUS       sequence-368073-        5212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses retron Eco1 ncRNA v35 (long a1/a2, barcode 8 [GCT-TGC]), 
            and Cas1 + Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5212)
  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 5212)
  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.016
FEATURES             Location/Qualifiers
     source          1..5212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             203..220
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        334..352
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    353..377
                     /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             1749..1793
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1831..1849)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1845..1892
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2122..2937)
                     /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(2122..2937)
                     /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     2845..2864
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2938..3029)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3045..3794)
                     /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(3055..3072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3295..3314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3990..5072)
                     /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     5034..5053
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5073..5150)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5196..5212,1..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 gaaaatggag
      121 aggtgcaatg cacctctcca cccgtttttt ctgacgtaag ggtgcgcata cggaatctta
      181 tcaactttca tgaaatccgc tgcatcacca tcatcaccac agccaggatc cgaattcgag
      241 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      301 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      361 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacaat
      421 gacctggctt ccccttaatc ccattccact caaagatcgc gtctccatga tctttctgca
      481 atatgggcag atcgatgtaa tagatggcgc gtttgtactt atcgacaaga cagggatccg
      541 cactcatatt cctgttggct cggttgcctg catcatgctg gaacctggta cacgggtttc
      601 gcatgcagct gtacgcctgg ctgcgcaagt tggaacattg ttggtatggg tgggggaagc
      661 gggcgttcgt gtttatgctt ctggtcagcc tggaggtgcg cgttcagata agctgctcta
      721 tcaggcaaaa cttgctctgg atgaagattt gcgtctgaag gtcgtacgta aaatgtttga
      781 acttcggttt ggagaacctg cgcctgcccg gcgctccgta gagcaactca gaggtataga
      841 aggcagtcgc gtgcgggcaa cctacgcact tctggcgaag caatacggcg tgacatggaa
      901 tggacgtcgc tacgatccga aagactggga aaagggcgat acgatcaacc aatgcattag
      961 cgctgcaact tcctgtttat acggcgtaac tgaagcggcg atacttgcag ctggttatgc
     1021 accagctatt gggtttgtgc atacaggaaa gcctctttcc tttgtttacg atattgcaga
     1081 catcattaaa tttgacactg ttgtaccgaa agcttttgag atagcgcgtc gtaaccctgg
     1141 tgagccggac cgggaagtcc gtttggcgtg cagggatatt tttcgcagta gtaaaacatt
     1201 agccaaattg attccgctta tagaggacgt gcttgccgct ggagaaatac aaccgccggc
     1261 cccacctgaa gatgcacagc ctgttgccat tccgcttcct gtttcactgg gagatgcagg
     1321 ccatcggagt agctgaaatg agtatgttgg tcgtggtcac tgaaaatgta cctcagcgct
     1381 tacgaggcag attagccatc tggttgttgg aggtacgtgc aggggtatat gtaggtgatg
     1441 tatccgcaaa aattcgtgaa atgatctggg aacaaatagc tggactggcg gaagaaggca
     1501 atgtagtgat ggcatgggca acgaatacag aaacgggatt tgagttccag acatttgggt
     1561 taaacaggcg taccccggta gatttggatg gtttaaggtt ggtgtctttt ttacctgttt
     1621 gagcggccgc actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     1681 ttatggcaga tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg
     1741 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     1801 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     1861 gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca
     1921 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     1981 tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact
     2041 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2101 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2161 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2221 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2281 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2341 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2401 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2461 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg
     2521 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2581 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2641 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2701 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2761 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2821 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     2881 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact
     2941 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3001 atttgaatgt atttagaaaa ataaacaaat aggcatgcag cgctcttccg cttcctcgct
     3061 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc
     3121 ggtaatacgg ttatccacag aatcagggga taaagccgga aagaacatgt gagcaaaaag
     3181 caaagcaccg gaagaagcca acgccgcagg cgtttttcca taggctccgc ccccctgacg
     3241 agcatcacaa aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga ctataaagat
     3301 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3361 ccggatacat gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3421 gttggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3481 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3541 gacacgactt atcgccactg gcagcagcca ttggtaactg atttagagga ctttgtcttg
     3601 aagttatgca cctgttaagg ctaaactgaa agaacagatt ttggtgagtg cggtcctcca
     3661 acccacttac cttggttcaa agagttggta gctcagcgaa ccttgagaaa accaccgttg
     3721 gtagcggtgg tttttcttta tttatgagat gatgaatcaa tcggtctatc aagtcaacga
     3781 acagctattc cgttactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
     3841 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
     3901 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     3961 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4021 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4081 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4141 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4201 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4261 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4321 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     4381 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     4441 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     4501 gaacttaatg ggcccgctaa cagcgcgatt tgcttgtgac ccaatgcgac cagatgctcc
     4561 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     4621 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     4681 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     4741 accaccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     4801 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     4861 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     4921 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     4981 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5041 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5101 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5161 acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta ta
//
