LOCUS       sequence-198325-        5351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas1, Cas2 under PLTetO-1 promoter, and TetR and LacI 
            constitutively..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5351)
  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 5351)
  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
FEATURES             Location/Qualifiers
     source          1..5351
                     /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."
     CDS             2608..3690
                     /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     complement(2627..2646)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      complement(3901..4489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3981..4000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4577..4671)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4692..5351)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5266..5285
                     /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 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     2821 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     2881 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     2941 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     3001 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     3061 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     3121 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     3181 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     3241 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     3301 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     3361 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     3421 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     3481 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     3541 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     3601 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3661 caggtttccc gactggaaag cgggcagtga gctttctaac taaaaaggcc tcccaaatcg
     3721 gggggccttt tttattgata acaaaacgct cctaggcgtt cggctgcggc gagcggtatc
     3781 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3841 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3901 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3961 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4021 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4081 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4141 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4201 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4261 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4321 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4381 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4441 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4501 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4561 catgactagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga
     4621 acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc
     4681 tcgatatcaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     4741 attctgccga catggaagcc atcacagacg gcatgatgaa cctgaatcgc cagcggcatc
     4801 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     4861 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     4921 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     4981 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     5041 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     5101 atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt catcaggcgg
     5161 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     5221 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     5281 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     5341 tttttctcca t
//
