LOCUS       sequence-367495-        5211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses retron Eco1 ncRNA v35 (long a1/a2, barcode 3 [CTG-CAG]), 
            and Cas1 + Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5211)
  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 5211)
  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.011
FEATURES             Location/Qualifiers
     source          1..5211
                     /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(2121..2936)
                     /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(2121..2936)
                     /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     2844..2863
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2937..3028)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3044..3793)
                     /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(3054..3071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3294..3313)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3989..5071)
                     /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     5033..5052
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5072..5149)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5195..5211,1..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 aggtctgaca gacctctcca 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 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2101 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     2161 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     2221 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     2281 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     2341 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     2401 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     2461 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga
     2521 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2581 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2641 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2701 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2761 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2821 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2881 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc
     2941 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3001 tttgaatgta tttagaaaaa taaacaaata ggcatgcagc gctcttccgc ttcctcgctc
     3061 actgactcgc tacgctcggt cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg
     3121 gtaatacggt tatccacaga atcaggggat aaagccggaa agaacatgtg agcaaaaagc
     3181 aaagcaccgg aagaagccaa cgccgcaggc gtttttccat aggctccgcc cccctgacga
     3241 gcatcacaaa aatcgacgct caagccagag gtggcgaaac ccgacaggac tataaagata
     3301 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3361 cggatacatg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3421 ttggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3481 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3541 acacgactta tcgccactgg cagcagccat tggtaactga tttagaggac tttgtcttga
     3601 agttatgcac ctgttaaggc taaactgaaa gaacagattt tggtgagtgc ggtcctccaa
     3661 cccacttacc ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg
     3721 tagcggtggt ttttctttat ttatgagatg atgaatcaat cggtctatca agtcaacgaa
     3781 cagctattcc gttactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     3841 tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa
     3901 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3961 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4021 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4081 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4141 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4201 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4261 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4321 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4381 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4441 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4501 aacttaatgg gcccgctaac agcgcgattt gcttgtgacc caatgcgacc agatgctcca
     4561 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4621 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4681 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4741 ccaccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4801 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4861 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4921 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4981 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5041 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5101 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5161 cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat a
//
