LOCUS       sequence-178394-        5226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial co-expression of folA (DHFR) and thyA(TS) with a barcode 
            within the noncoding region between the genes .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5226)
  AUTHORS   Schober AF, Mathis AD, Ingle C, Park JO, Chen L, Rabinowitz JD, 
            Junier I, Rivoire O, Reynolds KA
  TITLE     A Two-Enzyme Adaptive Unit within Bacterial Folate Metabolism.
  JOURNAL   Cell Rep. 2019 Jun 11;27(11):3359-3370.e7. doi: 
            10.1016/j.celrep.2019.05.030.
  PUBMED    31189117
REFERENCE   2  (bases 1 to 5226)
  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: 
            "10.1016/j.celrep.2019.05"; journalName: "Cell Rep"; date: 
            "2019-06-11- 11"; volume: "27"; issue: "11"; pages: "3359-3370.e7"
COMMENT     Sequence Label: pACYCDuet_WTEcoliDHFR_R166QTS
FEATURES             Location/Qualifiers
     source          1..5226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             12..56
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(94..112)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      108..155
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(376..1035)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     950..969
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1036..1138)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1530..1547)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1664..2209)
                     /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(1744..1763)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2434..3516)
                     /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     3478..3497
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3517..3594)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3640..3658
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3659..3683
                     /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             3739..3756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3769..4248
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     promoter        4333..4351
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4352..4376
                     /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)."
ORIGIN
        1 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
       61 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
      121 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
      181 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
      241 ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc
      301 attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata
      361 actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
      421 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
      481 catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg gggcgaagaa
      541 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
      601 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
      661 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
      721 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
      781 ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag
      841 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
      901 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
      961 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     1021 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     1081 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     1141 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     1201 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg
     1261 tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt
     1321 ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa
     1381 gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga
     1441 gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag
     1501 aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     1561 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg
     1621 aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc
     1681 cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac
     1741 tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct
     1801 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
     1861 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
     1921 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
     1981 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
     2041 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
     2101 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
     2161 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
     2221 tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc
     2281 tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac
     2341 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     2401 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2461 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2521 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     2581 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     2641 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     2701 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     2761 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     2821 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     2881 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     2941 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3001 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3061 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3121 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     3181 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     3241 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     3301 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     3361 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     3421 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     3481 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     3541 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     3601 ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg
     3661 aattgtgagc ggataacaat tcccctgtag aaataatttt gtttaacttt aataaggaga
     3721 tataccatgg gcagcagcca tcaccatcat caccacagcc aggatccgat gatcagtctg
     3781 attgcggcgt tagcggtaga tcgcgttatc ggcatggaaa acgccatgcc gtggaacctg
     3841 cctgccgatc tcgcctggtt taaacgcaac accttaaata aacccgtgat tatgggccgc
     3901 catacctggg aatcaatcgg tcgtccgttg ccaggacgca aaaatattat cctcagcagt
     3961 caaccgggta cggacgatcg cgtaacgtgg gtgaagtcgg tggatgaagc catcgcggcg
     4021 tgtggtgacg taccagaaat catggtgatt ggcggcggtc gcgtttatga acagttcttg
     4081 ccaaaagcgc aaaaactgta tctgacgcat atcgacgcag aagtggaagg cgacacccat
     4141 ttcccggatt acgagccgga tgactgggaa tcggtattca gcgaattcca cgatgctgat
     4201 gcgcagaact ctcacagcta ttgctttgag attctggagc ggcggtaaca ggcgtcgaca
     4261 agcttgcggc cgcgacagat gcttaagtcg aacagaaagt aatcgtattg tacacggccg
     4321 cataatcgaa attaatacga ctcactatag gggaattgtg agcggataac aattccccat
     4381 cttagtatat tagttaagta taagaaggag atatacatat ggcagatctc atgaaacagt
     4441 acctggagct gatgcaaaaa gttctggatg agggtaccca gaaaaacgac cgcacgggta
     4501 ccggaacgct gagcattttt ggccatcaga tgcgttttaa cctgcaggac ggattcccgc
     4561 tggttacgac caaacgctgc cacctgcgta gcattattca tgagctgctg tggtttctgc
     4621 aaggtgacac taacatcgcg tatctgcacg aaaacaatgt gacgatctgg gatgaatggg
     4681 ccgatgaaaa cggcgatctg ggcccagtgt atggtaaaca gtggcgcgcc tggccaacgc
     4741 cagatggccg ccacatcgat cagatcacga ccgtgctgaa tcaactgaaa aacgacccgg
     4801 acagccgccg cattattgtt tccgcgtgga atgtgggtga actggataaa atggcgctgg
     4861 cgccgtgcca tgcattcttc cagttttacg ttgcggacgg taagctgagc tgtcaacttt
     4921 atcagcagag ctgcgatgtt ttcctcggcc tgccgttcaa tatcgccagc tacgcgttac
     4981 tggtgcatat gatggcgcag cagtgcgacc tggaggtggg tgattttgtc tggaccggcg
     5041 gtgataccca cctgtacagc aaccatatgg accaaacgca tctgcaattg agccgtgagc
     5101 cgcgtccgct gccgaaactg attattaagc gcaaacccga gagcatcttt gactatcgtt
     5161 tcgaagactt cgagattgaa ggctacgatc cgcacccggg tatcaaagcg ccggtggcta
     5221 tctaac
//
