LOCUS       sequence-324205-        3714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pFA vector control expressing sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3714)
  AUTHORS   Flamholz AI, Dugan E, Blikstad C, Gleizer S, Ben-Nissan R, Amram S, 
            Antonovsky N, Ravishankar S, Noor E, Bar-Even A, Milo R, Savage D
  TITLE     Functional reconstitution of a bacterial CO2 concentrating mechanism
            in E. coli.
  JOURNAL   Elife. 2020 Oct 21;9. pii: 59882. doi: 10.7554/eLife.59882.
  PUBMED    33084575
REFERENCE   2  (bases 1 to 3714)
  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: "Elife."; 
            date: "2020-10-21"; volume: "9. pii: 59882. doi"; pages: " 
            10.7554/eLife.59882"
COMMENT     Sequence Label: pFA-sfGFP
FEATURES             Location/Qualifiers
     source          1..3714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(491..585)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(606..1265)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1180..1199
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1266..1368)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     1434..1452
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1473..1491,1492..1497,1498..1514,1515..1520,
                     1521..1546)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1473..1491
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1498..1516
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1556..1567
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1574..1576,1577..1579,1580..1582,1583..2293)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MSVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKD
                     DGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGI
                     KANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1622..1643)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2230..2251
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2300..2317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2379..2465
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2638..3261)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3169..3188
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3262..3366)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3412..3429)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3546..3714,1..377))
                     /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(3626..3645)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct
       61 ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg
      121 tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg
      181 atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt
      241 ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa
      301 ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca
      361 gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc tagatttcag
      421 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgttac
      481 tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
      541 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata
      601 tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
      661 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
      721 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
      781 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
      841 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
      901 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
      961 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     1021 agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga
     1081 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     1141 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     1201 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     1261 tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt
     1321 gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt
     1381 cgccagatat cgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1441 gtatcacgag gccctttcgt cttcacctcg agtccctatc agtgatagag attgacatcc
     1501 ctatcagtga tagagatact gagcacatca gcaggacgca ctgaccgaat tcattaaaga
     1561 ggagaaaggt accatgtccg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1621 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg gcgagggcga
     1681 gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1741 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1801 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1861 ggagcgcacc atctccttca aggacgacgg cacctacaag acccgcgccg aggtgaagtt
     1921 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1981 caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct atatcacggc
     2041 cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaacg tcgaggacgg
     2101 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2161 gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa
     2221 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2281 cgagctgtac aagagcggtc accatcacca tcaccattaa aatgtaactg tattcagcga
     2341 tgacgaaatt cttagctatt gtaatactct agaggcatca aataaaacga aaggctcagt
     2401 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     2461 caaatccgcc gccgctagca gttcgtagac ggaaacaaac gcagaatcca agcaaaaggg
     2521 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2581 catttatcag ggttatgtcc actgggttcg tgccttcatc cggatatcca ggaactgtta
     2641 agacccactt tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa
     2701 taagaaggct ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg
     2761 cggcatacta tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc
     2821 ttccaatacg caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc
     2881 tagtgaaaaa ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt
     2941 ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca
     3001 tctaaaactt ttagcgttat tacgtaaaaa atcttgccag ctttcccctt ctaaagggca
     3061 aaagtgagta tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt
     3121 attttttaca tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg
     3181 ggttgttaaa ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt
     3241 acttttatct aatctagaca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3301 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3361 tccgcgtttc cctcgacaat tcgcgctaga cctagggata tattccgctt cctcgctcac
     3421 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
     3481 agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag
     3541 ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag
     3601 tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc
     3661 gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atgg
//
