LOCUS       sequence-245545-        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_M42FG121VDHFR_WTTS
FEATURES             Location/Qualifiers
     source          1..5212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..29
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    30..54
                     /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             110..127
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             140..619
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIFGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEVDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     promoter        690..708
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    709..733
                     /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             1595..1639
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1677..1695)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1691..1738
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1959..2618)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2533..2552
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2619..2721)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3113..3130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3247..3792)
                     /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(3327..3346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4017..5099)
                     /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     5061..5080
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5100..5177)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 attaggaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta
       61 gaaataattt tgtttaactt taataaggag atataccatg ggcagcagcc atcaccatca
      121 tcaccacagc caggatccga tgatcagtct gattgcggcg ttagcggtag atcgcgttat
      181 cggcatggaa aacgccatgc cgtggaacct gcctgccgat ctcgcctggt ttaaacgcaa
      241 caccttaaat aaacccgtga ttttcggccg ccatacctgg gaatcaatcg gtcgtccgtt
      301 gccaggacgc aaaaatatta tcctcagcag tcaaccgggt acggacgatc gcgtaacgtg
      361 ggtgaagtcg gtggatgaag ccatcgcggc gtgtggtgac gtaccagaaa tcatggtgat
      421 tggcggcggt cgcgtttatg aacagttctt gccaaaagcg caaaaactgt atctgacgca
      481 tatcgacgca gaagtggaag tcgacaccca tttcccggat tacgagccgg atgactggga
      541 atcggtattc agcgaattcc acgatgctga tgcgcagaac tctcacagct attgctttga
      601 gattctggag cggcggtaac ctgtgcggcc gcataatgct taagtcgaac agaaagtaat
      661 cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc
      721 ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata tacatatggc
      781 agatctcatg aaacagtacc tggagctgat gcaaaaagtt ctggatgagg gtacccagaa
      841 aaacgaccgc acgggtaccg gaacgctgag catttttggc catcagatgc gttttaacct
      901 gcaggacgga ttcccgctgg ttacgaccaa acgctgccac ctgcgtagca ttattcatga
      961 gctgctgtgg tttctgcaag gtgacactaa catcgcgtat ctgcacgaaa acaatgtgac
     1021 gatctgggat gaatgggccg atgaaaacgg cgatctgggc ccagtgtatg gtaaacagtg
     1081 gcgcgcctgg ccaacgccag atggccgcca catcgatcag atcacgaccg tgctgaatca
     1141 actgaaaaac gacccggaca gccgccgcat tattgtttcc gcgtggaatg tgggtgaact
     1201 ggataaaatg gcgctggcgc cgtgccatgc attcttccag ttttacgttg cggacggtaa
     1261 gctgagctgt caactttatc agcgcagctg cgatgttttc ctcggcctgc cgttcaatat
     1321 cgccagctac gcgttactgg tgcatatgat ggcgcagcag tgcgacctgg aggtgggtga
     1381 ttttgtctgg accggcggtg atacccacct gtacagcaac catatggacc aaacgcatct
     1441 gcaattgagc cgtgagccgc gtccgctgcc gaaactgatt attaagcgca aacccgagag
     1501 catctttgac tatcgtttcg aagacttcga gattgaaggc tacgatccgc acccgggtat
     1561 caaagcgccg gtggctatct aactcgagtc tggtaaagaa accgctgctg cgaaatttga
     1621 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     1681 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1741 gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
     1801 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
     1861 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa
     1921 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca
     1981 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     2041 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     2101 atagtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     2161 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     2221 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     2281 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     2341 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg
     2401 aactccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     2461 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     2521 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     2581 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     2641 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
     2701 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg
     2761 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
     2821 tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg
     2881 tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga
     2941 cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg
     3001 gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa
     3061 aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca
     3121 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg
     3181 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa
     3241 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca
     3301 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct
     3361 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
     3421 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
     3481 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
     3541 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
     3601 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
     3661 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
     3721 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
     3781 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa
     3841 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca
     3901 tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     3961 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4021 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4081 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4141 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4201 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4261 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     4321 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4381 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4441 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4501 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4561 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4621 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4681 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4741 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4801 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     4861 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     4921 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     4981 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5041 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5101 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5161 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gc
//
