LOCUS       sequence-204272-        5315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas1, Cas2 under PLTetO-1 promoter, and TetR and LacI-GalS
            chimera constitutively..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5315)
  AUTHORS   Sheth RU, Yim SS, Wu FL, Wang HH
  TITLE     Multiplex recording of cellular events over time on CRISPR 
            biological tape.
  JOURNAL   Science. 2017 Dec 15;358(6369):1457-1461. doi: 
            10.1126/science.aao0958. Epub 2017 Nov 23.
  PUBMED    29170279
REFERENCE   2  (bases 1 to 5315)
  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: 
            "10.1126/science.aao0958"; journalName: "Science"; date: 
            "2017-12-15- 15"; volume: "358"; issue: "6369"; pages: "1457-1461"
COMMENT     Sequence Label: pRec-GalS
FEATURES             Location/Qualifiers
     source          1..5315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..103)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     169..187
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(208..226,227..232,233..249,250..255,256..281)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    208..226
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      1541..1612
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1628..1655
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1707..1741
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             1776..2402
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1852..1871)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      2408..2494
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        2530..2607
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(2627..2646)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      complement(3865..4453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3945..3964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4541..4635)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4656..5315)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5230..5249
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
       61 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
      121 gatatcgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
      181 acgaggccct ttcgtcttca cctcgagtcc ctatcagtga tagagattga catccctatc
      241 agtgatagag atactgagca catcagcagg acgcactgac cgtttaataa atatatacat
      301 atcaggacaa cgcctaatga cctggcttcc ccttaatccc attccactca aagatcgcgt
      361 ctccatgatc tttctgcaat atgggcagat cgatgtaata gatggcgcgt ttgtacttat
      421 cgacaagaca gggatccgca ctcatattcc tgttggctcg gttgcctgca tcatgctgga
      481 acctggtaca cgggtttcgc atgcagctgt acgcctggct gcgcaagttg gaacattgtt
      541 ggtatgggtg ggggaagcgg gcgttcgtgt ttatgcttct ggtcagcctg gaggtgcgcg
      601 ttcagataag ctgctctatc aggcaaaact tgctctggat gaagatttgc gtctgaaggt
      661 cgtacgtaaa atgtttgaac ttcggtttgg agaacctgcg cctgcccggc gctccgtaga
      721 gcaactcaga ggtatagaag gcagtcgcgt gcgggcaacc tacgcacttc tggcgaagca
      781 atacggcgtg acatggaatg gacgtcgcta cgatccgaaa gactgggaaa agggcgatac
      841 gatcaaccaa tgcattagcg ctgcaacttc ctgtttatac ggcgtaactg aagcggcgat
      901 acttgcagct ggttatgcac cagctattgg gtttgtgcat acaggaaagc ctctttcctt
      961 tgtttacgat attgcagaca tcattaaatt tgacactgtt gtaccgaaag cttttgagat
     1021 agcgcgtcgt aaccctggtg agccggaccg ggaagtccgt ttggcgtgca gggatatttt
     1081 tcgcagtagt aaaacattag ccaaattgat tccgcttata gaggacgtgc ttgccgctgg
     1141 agaaatacaa ccgccggccc cacctgaaga tgcacagcct gttgccattc cgcttcctgt
     1201 ttcactggga gatgcaggcc atcggagtag ctgaaatgag tatgttggtc gtggtcactg
     1261 aaaatgtacc tccgcgctta cgaggcagat tagccatctg gttgttggag gtacgtgcag
     1321 gggtatatgt aggtgatgta tccgcaaaaa ttcgtgaaat gatctgggaa caaatagctg
     1381 gactggcgga agaaggcaat gtagtgatgg catgggcaac gaatacggaa acgggatttg
     1441 agttccagac atttgggtta aacaggcgta ccccggtaga tttggatggt ttaaggttgg
     1501 tgtctttttt acctgtttga taataatcta gaccaggcat caaataaaac gaaaggctca
     1561 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     1621 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat aggtacccgt ggatcctcta
     1681 gagtcgacct gcaggcatgc aagcttttta cggctagctc agtcctaggt atagtgctag
     1741 cccagccaga gaaacactct ttaacagggg gtagtatgat gtctcgttta gataaaagta
     1801 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     1861 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     1921 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     1981 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     2041 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     2101 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     2161 agaatgcatt atatgcactc agcgcagtgg ggcattttac tttaggttgc gtattggaag
     2221 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     2281 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     2341 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtctt
     2401 aaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     2461 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ccctagacga agcggcatgc
     2521 atttacgttg acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg
     2581 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     2641 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     2701 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     2761 gtggcagcgc aactggcggg caaacagtcg gacaccattg gcgtggtggt gatggacgtt
     2821 tctgatgcgt ttttcggcgc gctggtaaaa gcggtggatc tggtcgctca gcagcatcag
     2881 aaatacgtgc taatcggcaa tagctatcat gaagcggaaa aagagcgtca cgccattgag
     2941 gtgttaattc gccagcgttg taatgcgttg attgttcact caaaagcatt gagtgacgat
     3001 gaactggcgc aatttatgga taacattccc ggtatggtgt taatcaaccg cgttgtgccg
     3061 gggtacgccc atcgttgcgt ttgcctggat aatctcagcg gtgcccgaat ggcgacgcgc
     3121 atgttgctga ataacggtca tcaacgtatt ggttatcttt cttccagcca cggcattgaa
     3181 gatgacgcca tgcgtaaagc aggctggatg agtgcgttga aagagcagga tattattccg
     3241 ccggaaagct ggattggcac tggtacgccg gacatgccgg gcggtgaggc ggcgatggtt
     3301 aaactgctgg ggcgcaatct acaacttacc gctgtatttg cttataacga caatatggcc
     3361 gctggcgcac tgacagcatt aaaagataat ggcattgcga ttccgttaca tctctcaatc
     3421 atcggtttcg atgatattcc catcgcccgt tacaccgacc cgcaattaac gaccgtgcgt
     3481 tatcccattg cttcaatggc gaaattagcc accgaactgg ccttgcaggg ggcagcaggc
     3541 aatattgatc ctcgtgccag ccactggttt atgccgacgt tagtgcgtcg ccattctgtc
     3601 gcaacacgcc agaatgcggc ggcgatcact aactcaacaa atcaggcgat gtaagctttc
     3661 taactaaaaa ggcctcccaa atcggggggc cttttttatt gataacaaaa cgctcctagg
     3721 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3781 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3841 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3901 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3961 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4021 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     4081 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4141 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4201 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4261 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4321 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4381 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4441 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4501 aaactcacgt taagggattt tggtcatgac tagtgcttgg attctcacca ataaaaaacg
     4561 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     4621 ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg ccactcatcg
     4681 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca gacggcatga
     4741 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     4801 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     4861 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
     4921 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     4981 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     5041 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgaaat
     5101 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     5161 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     5221 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     5281 tcaacggtgg tatatccagt gatttttttc tccat
//
