LOCUS       sequence-178383-        5212 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 5212)
  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 5212)
  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_G121VDHFR_WTTS
FEATURES             Location/Qualifiers
     source          1..5212
                     /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(1333..1992)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1907..1926
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1993..2095)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2487..2504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2621..3166)
                     /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(2701..2720)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3391..4473)
                     /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     4435..4454
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4474..4551)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4597..4615
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4616..4640
                     /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             4696..4713
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4726..5205
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     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 gcaaccaggc gtttaagggc accaataact
     1321 gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag
     1381 cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat
     1441 cagcaccttg tcgccttgcg tataatattt gcccatagtg aaaacggggg cgaagaagtt
     1501 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac
     1561 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc
     1621 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag
     1681 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca
     1741 tatcaccagc tcaccgtctt tcattgccat acggaactcc ggatgagcat tcatcaggcg
     1801 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa
     1861 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa
     1921 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat
     1981 ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc
     2041 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc
     2101 tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac caggatttat
     2161 ttattctgcg aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga
     2221 tgctgccaac ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg
     2281 tttctatcag ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca
     2341 ccgccggaca tcagcgctag cggagtgtat actggcttac tatgttggca ctgatgaggg
     2401 tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat
     2461 atgtgataca ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     2521 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
     2581 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc
     2641 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     2701 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     2761 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     2821 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     2881 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     2941 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     3001 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     3061 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     3121 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     3181 taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     3241 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     3301 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3361 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3421 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3481 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3541 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3601 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3661 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3721 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3781 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3841 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     3901 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     3961 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4021 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4081 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4141 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4201 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4261 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4321 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4381 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4441 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4501 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4561 gacgctctcc cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat
     4621 tgtgagcgga taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat
     4681 accatgggca gcagccatca ccatcatcac cacagccagg atccgatgat cagtctgatt
     4741 gcggcgttag cggtagatcg cgttatcggc atggaaaacg ccatgccgtg gaacctgcct
     4801 gccgatctcg cctggtttaa acgcaacacc ttaaataaac ccgtgattat gggccgccat
     4861 acctgggaat caatcggtcg tccgttgcca ggacgcaaaa atattatcct cagcagtcaa
     4921 ccgggtacgg acgatcgcgt aacgtgggtg aagtcggtgg atgaagccat cgcggcgtgt
     4981 ggtgacgtac cagaaatcat ggtgattggc ggcggtcgcg tttatgaaca gttcttgcca
     5041 aaagcgcaaa aactgtatct gacgcatatc gacgcagaag tggaagtcga cacccatttc
     5101 ccggattacg agccggatga ctgggaatcg gtattcagcg aattccacga tgctgatgcg
     5161 cagaactctc acagctattg ctttgagatt ctggagcggc ggtaacataa gc
//
