LOCUS       sequence-378516-        5180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial co-expression of E. coli folA (Dihydrofolate Reductase) 
            and E. coli thyA (Thymidylate Synthase).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5180)
  AUTHORS   Nguyen TN, Ingle C, Thompson S, Reynolds KA
  TITLE     The genetic landscape of a metabolic interaction.
  JOURNAL   Nat Commun. 2024 Apr 18;15(1):3351. doi: 10.1038/s41467-024-47671-0.
  PUBMED    38637543
REFERENCE   2  (bases 1 to 5180)
  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"; journalName: "Nat 
            Commun."; date: "2024-04-18"; volume: "15(1):3351. doi"; pages: " 
            10.1038/s41467-024-47671-0"
COMMENT     Sequence Label: pTETDuet_D27NDHFR_R127ATS
FEATURES             Location/Qualifiers
     source          1..5180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(34..52,53..58,59..75,76..81,82..107)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    34..52
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    59..77
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             117..128
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             956..1000
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1038..1056)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1319..1978)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1893..1912
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1979..2081)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2473..2490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2607..3152)
                     /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(2687..2706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3377..4459)
                     /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     4421..4440
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4460..4537)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4583..4602
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4583..4601
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4602..4626
                     /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             4670..5149
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPANLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
ORIGIN
        1 cttaagtcga acagaaagta atcgtattgt acatccctat cagtgataga gattgacatc
       61 cctatcagtg atagagatac tgagcacatc agcaggacgc actgaccgaa ttcattaaag
      121 aggagaaagg taccatgctg cagatctcat gaaacagtac ctggagctga tgcaaaaagt
      181 tctggatgag ggtacccaga aaaacgaccg cacgggtacc ggaacgctga gcatttttgg
      241 ccatcagatg cgttttaacc tgcaggacgg attcccgctg gttacgacca aacgctgcca
      301 cctgcgtagc attattcatg agctgctgtg gtttctgcaa ggtgacacta acatcgcgta
      361 tctgcacgaa aacaatgtga cgatctggga tgaatgggcc gatgaaaacg gcgatctggg
      421 cccagtgtat ggtaaacagt ggcgcgcctg gccaacgcca gatggccgcc acatcgatca
      481 gatcacgacc gtgctgaatc aactgaaaaa cgacccggac agccgcgcca ttattgtttc
      541 cgcgtggaat gtgggtgaac tggataaaat ggcgctggcg ccgtgccatg cattcttcca
      601 gttttacgtt gcggacggta agctgagctg tcaactttat cagcgcagct gcgatgtttt
      661 cctcggcctg ccgttcaata tcgccagcta cgcgttactg gtgcatatga tggcgcagca
      721 gtgcgacctg gaggtgggtg attttgtctg gaccggcggt gatacccacc tgtacagcaa
      781 ccatatggac caaacgcatc tgcaattgag ccgtgagccg cgtccgctgc cgaaactgat
      841 tattaagcgc aaacccgaga gcatctttga ctatcgtttc gaagacttcg agattgaagg
      901 ctacgatccg cacccgggta tcaaagcgcc ggtggctatc taactcgagt ctggtaaaga
      961 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     1021 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1081 ggtcttgagg ggtttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt
     1141 ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc
     1201 cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc cgcttattat
     1261 cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataactgcct taaaaaaatt
     1321 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     1381 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     1441 cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc atattggcca
     1501 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     1561 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     1621 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     1681 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     1741 cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca agaatgtgaa
     1801 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     1861 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     1921 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt
     1981 tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta
     2041 tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa
     2101 agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt
     2161 gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac
     2221 tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctgt
     2281 ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag
     2341 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc
     2401 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat
     2461 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     2521 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     2581 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     2641 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     2701 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc
     2761 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc
     2821 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt
     2881 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc
     2941 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa
     3001 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt
     3061 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc
     3121 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat
     3181 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     3241 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     3301 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     3361 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3421 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3481 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     3541 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     3601 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     3661 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     3721 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     3781 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     3841 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     3901 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     3961 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4021 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4081 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4141 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4201 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     4261 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     4321 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     4381 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     4441 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4501 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     4561 tgcgactcct gcattaggaa attaatacga ctcactatag gggaattgtg agcggataac
     4621 aattcccctg tagaaataat tttgtttaac tttaataagg agatatacca tgatcagtct
     4681 gattgcggcg ttagcggtag atcgcgttat cggcatggaa aacgccatgc cgtggaacct
     4741 gcctgccaac ctcgcctggt ttaaacgcaa caccttaaat aaacccgtga ttatgggccg
     4801 ccatacctgg gaatcaatcg gtcgtccgtt gccaggacgc aaaaatatta tcctcagcag
     4861 tcaaccgggt acggacgatc gcgtaacgtg ggtgaagtcg gtggatgaag ccatcgcggc
     4921 gtgtggtgac gtaccagaaa tcatggtgat tggcggcggt cgcgtttatg aacagttctt
     4981 gccaaaagcg caaaaactgt atctgacgca tatcgacgca gaagtggaag gcgacaccca
     5041 tttcccggat tacgagccgg atgactggga atcggtattc agcgaattcc acgatgctga
     5101 tgcgcagaac tctcacagct attgctttga gattctggag cggcggtaac aggcgtcgac
     5161 aagcttgcgg ccgcataatg
//
