LOCUS       sequence-324171-        3871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pFE vector control expressing sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3871)
  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 3871)
  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: pFE-sfGFP
FEATURES             Location/Qualifiers
     source          1..3871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(236..859)
                     /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     767..786
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(860..964)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1140..1728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1220..1239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1816..1910)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1941..2735)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2052..2071)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2662..2681
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2914..2932
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2953..2971,2972..2977,2978..2994,2995..3000,
                     3001..3026)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2953..2971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2978..2996
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             3036..3047
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(3054..3056,3057..3059,3060..3062,3063..3773)
                     /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(3102..3123)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3710..3731
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3780..3797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      join(3848..3871,1..63)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
       61 aatccgccgc cgctagcagt tcgtagacgg aaacaaacgc agaatccaag caaaagggaa
      121 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      181 tttatcaggg ttatgtccac tgggttcgtg ccttcatccg gatatccagg aactgttaag
      241 acccactttc acatttaagt tgtttttcta atccgcatat gatcaattca aggccgaata
      301 agaaggctgg ctctgcacct tggtgatcaa ataattcgat agcttgtcgt aataatggcg
      361 gcatactatc agtagtaggt gtttcccttt cttctttagc gacttgatgc tcttgatctt
      421 ccaatacgca acctaaagta aaatgcccca cagcgctgag tgcatataat gcattctcta
      481 gtgaaaaacc ttgttggcat aaaaaggcta attgattttc gagagtttca tactgttttt
      541 ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg atgacttagt aaagcacatc
      601 taaaactttt agcgttatta cgtaaaaaat cttgccagct ttccccttct aaagggcaaa
      661 agtgagtatg gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa gcccgcttat
      721 tttttacatg ccaatacaat gtaggctgct ctacacctag cttctgggcg agtttacggg
      781 ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta atcactttac
      841 ttttatctaa tctagacata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      961 cgcgtttccc tcgacaattc gcgctagacc tagggcgttc ggctgcggcg agcggtatca
     1021 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1081 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1141 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1201 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1261 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1321 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1381 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1441 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1501 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1561 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1621 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1681 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1741 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1801 atgactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
     1861 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct
     1921 ctcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     1981 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     2041 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     2101 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     2161 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     2221 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     2281 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     2341 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     2401 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     2461 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     2521 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     2581 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     2641 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     2701 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     2761 tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
     2821 actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cgacgtctaa
     2881 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     2941 cttcacctcg agtccctatc agtgatagag attgacatcc ctatcagtga tagagatact
     3001 gagcacatca gcaggacgca ctgaccgaat tcattaaaga ggagaaaggt accatgtccg
     3061 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     3121 acgtaaacgg ccacaagttc agcgtgcgcg gcgagggcga gggcgatgcc accaacggca
     3181 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     3241 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     3301 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atctccttca
     3361 aggacgacgg cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     3421 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     3481 tggagtacaa cttcaacagc cacaacgtct atatcacggc cgacaagcag aagaacggca
     3541 tcaaggcgaa cttcaagatc cgccacaacg tcgaggacgg cagcgtgcag ctcgccgacc
     3601 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     3661 tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3721 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagagcggtc
     3781 accatcacca tcaccattaa gaattcctgc agcccggggg atcccatggt acgcgtgcta
     3841 gaggcatcaa ataaaacgaa aggctcagtc g
//
