LOCUS       sequence-367485-        6077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Eco1 RT and Cas1 + Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6077)
  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 6077)
  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.079
FEATURES             Location/Qualifiers
     source          1..6077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=BBa_J23115
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.15"
     RBS             43..54
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1040..1057
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     1171..1190
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1171..1189
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1190..1214
                     /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             2586..2630
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2668..2686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2682..2729
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(2959..3750,3751..3819))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3582..3601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3820..3911)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3927..4676)
                     /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(3937..3954)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4177..4196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4872..5954)
                     /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     5916..5935
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5955..6032)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tttatagcta gctcagccct tggtacaatg ctagctctag agaaagagga gaaatactag
       61 atgaaatccg ctgaatattt gaacactttt agattgagaa atctcggcct acctgtcatg
      121 aacaatttgc atgacatgtc taaggcgact cgcatatctg ttgaaacact tcggttgtta
      181 atctatacag ctgattttcg ctataggatc tacactgtag aaaagaaagg cccagagaag
      241 agaatgagaa ccatttacca accttctcga gaacttaaag ccttacaagg atgggttcta
      301 cgtaacattt tagataaact gtcgtcatct cctttttcta ttggatttga aaagcaccaa
      361 tctattttga ataatgctac cccgcatatt ggggcaaact ttatactgaa tattgatttg
      421 gaggattttt tcccaagttt aactgctaac aaagtttttg gagtgttcca ttctcttggt
      481 tataatcgac taatatcttc agttttgaca aaaatatgtt gttataaaaa tctgctacca
      541 caaggtgctc catcatcacc taaattagct aatctaatat gttctaaact tgattatcgt
      601 attcagggtt atgcaggtag tcggggcttg atatatacga gatatgccga tgatctcacc
      661 ttatctgcac agtctatgaa aaaggttgtt aaagcacgtg attttttatt ttctataatc
      721 ccaagtgaag gattggttat taactcaaaa aaaacttgta ttagtgggcc tcgtagtcag
      781 aggaaagtta caggtttagt tatttcacaa gagaaagttg ggataggtag agaaaaatat
      841 aaagaaatta gagcaaagat acatcatata ttttgcggta agtcttctga gatagaacac
      901 gttaggggat ggttgtcatt tattttaagt gtggattcaa aaagccatag gagattaata
      961 acttatatta gcaaattaga aaaaaaatat ggaaagaacc ctttaaataa agcgaagacc
     1021 taatgacatg ggcagcagcc atcaccatca tcaccacagc caggatccga attcgagctc
     1081 ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa
     1141 tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag
     1201 cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat atacaatgac
     1261 ctggcttccc cttaatccca ttccactcaa agatcgcgtc tccatgatct ttctgcaata
     1321 tgggcagatc gatgtaatag atggcgcgtt tgtacttatc gacaagacag ggatccgcac
     1381 tcatattcct gttggctcgg ttgcctgcat catgctggaa cctggtacac gggtttcgca
     1441 tgcagctgta cgcctggctg cgcaagttgg aacattgttg gtatgggtgg gggaagcggg
     1501 cgttcgtgtt tatgcttctg gtcagcctgg aggtgcgcgt tcagataagc tgctctatca
     1561 ggcaaaactt gctctggatg aagatttgcg tctgaaggtc gtacgtaaaa tgtttgaact
     1621 tcggtttgga gaacctgcgc ctgcccggcg ctccgtagag caactcagag gtatagaagg
     1681 cagtcgcgtg cgggcaacct acgcacttct ggcgaagcaa tacggcgtga catggaatgg
     1741 acgtcgctac gatccgaaag actgggaaaa gggcgatacg atcaaccaat gcattagcgc
     1801 tgcaacttcc tgtttatacg gcgtaactga agcggcgata cttgcagctg gttatgcacc
     1861 agctattggg tttgtgcata caggaaagcc tctttccttt gtttacgata ttgcagacat
     1921 cattaaattt gacactgttg taccgaaagc ttttgagata gcgcgtcgta accctggtga
     1981 gccggaccgg gaagtccgtt tggcgtgcag ggatattttt cgcagtagta aaacattagc
     2041 caaattgatt ccgcttatag aggacgtgct tgccgctgga gaaatacaac cgccggcccc
     2101 acctgaagat gcacagcctg ttgccattcc gcttcctgtt tcactgggag atgcaggcca
     2161 tcggagtagc tgaaatgagt atgttggtcg tggtcactga aaatgtacct ccgcgcttac
     2221 gaggcagatt agccatctgg ttgttggagg tacgtgcagg ggtatatgta ggtgatgtat
     2281 ccgcaaaaat tcgtgaaatg atctgggaac aaatagctgg actggcggaa gaaggcaatg
     2341 tagtgatggc atgggcaacg aatacagaaa cgggatttga gttccagaca tttgggttaa
     2401 acaggcgtac cccggtagat ttggatggtt taaggttggt gtctttttta cctgtttgag
     2461 cggccgcact cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatta
     2521 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
     2581 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
     2641 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2701 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
     2761 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
     2821 aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt
     2881 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2941 ccgctcatga attaattctt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3001 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3061 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3121 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3181 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3241 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3301 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3361 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3421 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3481 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3541 tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag
     3601 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3661 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3721 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3781 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3841 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3901 aaataaacaa ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc
     3961 ggtcgttcga ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac
     4021 agaatcaggg gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc
     4081 caacgccgca ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4141 gctcaagcca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4201 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac atgtccgcct
     4261 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg
     4321 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4381 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4441 tggcagcagc cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa
     4501 ggctaaactg aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc
     4561 aaagagttgg tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt
     4621 tatttatgag atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc
     4681 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     4741 aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4801 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4861 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4921 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4981 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5041 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5101 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
     5161 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5221 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5281 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5341 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5401 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5461 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5521 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5581 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5641 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5701 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5761 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5821 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5881 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5941 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6001 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6061 ctcctgcatt aggaaat
//
