LOCUS       sequence-382527-        4475 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Contains a RamR-regulated promoter expressing GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4475)
  AUTHORS   d'Oelsnitz S, Kim W, Burkholder NT, Javanmardi K, Thyer R, Zhang Y, 
            Alper HS, Ellington AD
  TITLE     Using fungible biosensors to evolve improved alkaloid biosyntheses.
  JOURNAL   Nat Chem Biol. 2022 Jul 7. pii: 10.1038/s41589-022-01072-w. doi: 
            10.1038/s41589-022-01072-w.
  PUBMED    35799063
REFERENCE   2  (bases 1 to 4475)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2022 Jul 7. pii: 10.1038/s41589-022-01072-w. doi: 
            10.1038/s41589-022-01072-w."
COMMENT     Sequence Label: Pramr-GFP
FEATURES             Location/Qualifiers
     source          1..4475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(178..1260)
                     /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     1222..1241
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1261..1338)
                     /gene="lacI"
                     /label=lacI promoter
     RBS             1559..1570
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1607..2320
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1637..1665)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2260..2280
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(2595..3254)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3169..3188
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3255..3357)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3749..3766)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3883..4428)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3963..3982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc
       61 atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      121 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      181 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      241 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      301 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      361 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
      421 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
      481 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
      541 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
      601 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
      661 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
      721 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      781 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      841 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      901 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      961 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1021 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1081 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1141 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1201 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1261 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1321 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1381 aattataaac gcaggtcttt tcgccggtgt atttcttgca aaaaaaaccc cgccgaagcg
     1441 gggttttttt ttttacaggg ctataaggta gatccgggtc gacctttgaa aagtaccttg
     1501 acagcgtatc tttgctttct ataatgagtg cttactcact cataatcaag tactagagaa
     1561 agaggagaaa tactagatgg ggatgaccat gattacgcca agctttatga gtaaaggaga
     1621 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
     1681 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa
     1741 atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactttcgc
     1801 gtatggtctt caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa
     1861 gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
     1921 ctacaagaca cgtgctgaag tcaaatttga aggtgatacc cttgttaata gaatcgagtt
     1981 aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta
     2041 taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt
     2101 caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa
     2161 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc
     2221 tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
     2281 agctgctggg attacacatg gcatggatga actatacaaa taataaagag agaatataaa
     2341 aagccagatt attaatccgg cttttttatt atttctgaaa cctcaggcat ttgagaagca
     2401 cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc
     2461 tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga atttctgcca
     2521 ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg gcaccaataa
     2581 ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg taattcatta
     2641 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
     2701 atcagcacct tgtcgccttg cgtataatat ttgcccatag tgaaaacggg ggcgaagaag
     2761 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
     2821 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
     2881 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
     2941 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     3001 catatcacca gctcaccgtc tttcattgcc atacggaact ccggatgagc attcatcagg
     3061 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     3121 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     3181 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     3241 atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg
     3301 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg
     3361 tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt
     3421 atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt
     3481 gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc
     3541 tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta acggcaaaag
     3601 caccgccgga catcagcgct agcggagtgt atactggctt actatgttgg cactgatgag
     3661 ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga
     3721 atatgtgata caggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     3781 gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga
     3841 agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc
     3901 ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact
     3961 ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt
     4021 tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac
     4081 tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt
     4141 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa
     4201 aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc
     4261 gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta
     4321 cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt
     4381 ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt
     4441 attaatcaga taaaatattt ctagatttca gtgca
//
