LOCUS       sequence-175713-        5210 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 5210)
  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 5210)
  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_M42FG121VDHFR_WTTS
FEATURES             Location/Qualifiers
     source          1..5210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..82
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    83..107
                     /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             969..1013
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1051..1069)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1065..1112
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1332..1991)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1906..1925
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1992..2094)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2486..2503)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2620..3164)
                     /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(2700..2719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3389..4471)
                     /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     4433..4452
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4472..4549)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4595..4613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4614..4638
                     /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             4694..4711
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4724..5203
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIFGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEVDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
ORIGIN
        1 ggccgcataa tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga
       61 aattaatacg actcactata ggggaattgt gagcggataa caattcccca tcttagtata
      121 ttagttaagt ataagaagga gatatacata tggcagatct catgaaacag tacctggagc
      181 tgatgcaaaa agttctggat gagggtaccc agaaaaacga ccgcacgggt accggaacgc
      241 tgagcatttt tggccatcag atgcgtttta acctgcagga cggattcccg ctggttacga
      301 ccaaacgctg ccacctgcgt agcattattc atgagctgct gtggtttctg caaggtgaca
      361 ctaacatcgc gtatctgcac gaaaacaatg tgacgatctg ggatgaatgg gccgatgaaa
      421 acggcgatct gggcccagtg tatggtaaac agtggcgcgc ctggccaacg ccagatggcc
      481 gccacatcga tcagatcacg accgtgctga atcaactgaa aaacgacccg gacagccgcc
      541 gcattattgt ttccgcgtgg aatgtgggtg aactggataa aatggcgctg gcgccgtgcc
      601 atgcattctt ccagttttac gttgcggacg gtaagctgag ctgtcaactt tatcagcgca
      661 gctgcgatgt tttcctcggc ctgccgttca atatcgccag ctacgcgtta ctggtgcata
      721 tgatggcgca gcagtgcgac ctggaggtgg gtgattttgt ctggaccggc ggtgataccc
      781 acctgtacag caaccatatg gaccaaacgc atctgcaatt gagccgtgag ccgcgtccgc
      841 tgccgaaact gattattaag cgcaaacccg agagcatctt tgactatcgt ttcgaagact
      901 tcgagattga aggctacgat ccgcacccgg gtatcaaagc gccggtggct atctaactcg
      961 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     1021 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     1081 gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca
     1141 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     1201 tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt
     1261 catccgctta ttatcactta ttcaggcgta gcaccaggcg tttaagggca ccaataactg
     1321 ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     1381 attctgccga catggaagcc atcacagacg gcatgatgaa cctgaatcgc cagcggcatc
     1441 agcaccttgt cgccttgcgt ataatatttg cccatagtga aaacgggggc gaagaagttg
     1501 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     1561 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     1621 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     1681 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     1741 atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg
     1801 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     1861 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     1921 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     1981 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
     2041 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct
     2101 cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt
     2161 tattctgcga agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat
     2221 gctgccaact tactgattta gtgtatgatg gtgtttttga ggtgctccag tggcttctgt
     2281 ttctatcagc tgtccctcct gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac
     2341 cgccggacat cagcgctagc ggagtgtata ctggcttact atgttggcac tgatgagggt
     2401 gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt cagcagaata
     2461 tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac
     2521 tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga
     2581 tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc
     2641 tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata
     2701 aagataccag gcgtttcccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg
     2761 tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag
     2821 ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga
     2881 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc
     2941 accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg
     3001 gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc
     3061 ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt
     3121 cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta
     3181 atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     3241 tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa
     3301 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3361 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3421 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3481 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3541 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     3601 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     3661 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     3721 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     3781 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     3841 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     3901 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     3961 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4021 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4081 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4141 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4201 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4261 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4321 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4381 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     4441 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4501 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4561 cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat aggggaattg
     4621 tgagcggata acaattcccc tgtagaaata attttgttta actttaataa ggagatatac
     4681 catgggcagc agccatcacc atcatcacca cagccaggat ccgatgatca gtctgattgc
     4741 ggcgttagcg gtagatcgcg ttatcggcat ggaaaacgcc atgccgtgga acctgcctgc
     4801 cgatctcgcc tggtttaaac gcaacacctt aaataaaccc gtgattttcg gccgccatac
     4861 ctgggaatca atcggtcgtc cgttgccagg acgcaaaaat attatcctca gcagtcaacc
     4921 gggtacggac gatcgcgtaa cgtgggtgaa gtcggtggat gaagccatcg cggcgtgtgg
     4981 tgacgtacca gaaatcatgg tgattggcgg cggtcgcgtt tatgaacagt tcttgccaaa
     5041 agcgcaaaaa ctgtatctga cgcatatcga cgcagaagtg gaagtcgaca cccatttccc
     5101 ggattacgag ccggatgact gggaatcggt attcagcgaa ttccacgatg ctgatgcgca
     5161 gaactctcac agctattgct ttgagattct ggagcggcgg taacctgtgc
//
