LOCUS       sequence-492574-        6096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of ECFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6096)
  AUTHORS   Abrams RPM, Donahue RG, Ma J, Mao Y, Diolaiti ME, Ashworth A
  TITLE     An engineered cysteine sensor optimized for high-throughput 
            screening identifies regulators of intracellular thiol levels.
  JOURNAL   Cell Chem Biol. 2025 Nov 10:S2451-9456(25)00343-5. doi: 
            10.1016/j.chembiol.2025.10.006.
  PUBMED    41218609
REFERENCE   2  (bases 1 to 6096)
  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: "Cell Chem 
            Biol. 2025 Nov 10:S2451-9456(25)00343-5. doi: 
            10.1016/j.chembiol.2025.10.006."
COMMENT     Sequence Label: pcDNA3.1_ECFP
FEATURES             Location/Qualifiers
     source          1..6096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1229..1248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1687..1704)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1693..1917
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1963..2391
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2050..2069)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2260..2281
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2400..2420)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2405..2734
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2585..2720
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2647..2666
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2801..3595
                     /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(2855..2874)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3465..3484
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3769..3890
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3806..3825)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3860..3879
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3939..3955)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3939..3955)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3952..3974)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3963..3979
                     /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)."
     promoter        complement(join(3987..3993,3994..4011,4012..4017))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4032..4053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4170..4187)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4341..4929)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4421..4440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5100..5891,5892..5960))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5723..5742
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5961..6065)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggtttc caagggcgaa gaactgttca ccggcgtggt
      961 gcccattctg gtggaactgg acggggatgt gaacggccac aagtttagcg ttagcggcga
     1021 aggcgaaggg gatgccacat acggaaagct gaccctgaag ttcatctgca ccaccggcaa
     1081 gctgcctgtg ccttggccta cactggtcac cacactgaca tggggcgtgc agtgcttcag
     1141 cagatacccc gaccatatga agcagcacga cttcttcaag agcgccatgc ctgagggcta
     1201 cgtgcaagag cggaccatct tctttaagga cgacggcaac tacaagacca gggccgaagt
     1261 gaagttcgag ggcgacaccc tggtcaaccg gatcgagctg aagggcatcg acttcaaaga
     1321 ggacggcaac atcctgggcc acaagctcga gtacaactac atcagccaca acgtgtacat
     1381 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccggc acaacatcga
     1441 ggacggctct gtgcagctgg ccgatcacta ccagcagaac acacccatcg gagatggccc
     1501 tgtgctgctg cccgataacc actacctgag cacacagagc gccctgagca aggaccccaa
     1561 cgagaagagg gatcacatgg tgctgctgga attcgtgacc gccgctggca tcacactcgg
     1621 catggacgaa ctgtacaaat gagcggccgc tcgagtctag agggcccgtt taaacccgct
     1681 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1741 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1801 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1861 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     1921 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     1981 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2041 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2101 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2161 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2221 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2281 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2341 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     2401 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2461 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2521 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2581 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2641 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     2701 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     2761 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2821 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2881 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     2941 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3001 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3061 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3121 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3181 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3241 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3301 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3361 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3421 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3481 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3541 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3601 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3661 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3721 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     3781 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3841 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     3901 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     3961 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4021 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4081 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4141 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4201 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4261 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4321 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4381 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4441 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4501 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4561 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4621 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4681 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4741 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4801 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4861 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4921 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4981 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5041 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5101 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5161 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5221 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5281 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5341 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5401 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5461 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5521 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5581 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5641 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5701 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5761 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5821 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5881 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5941 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6001 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6061 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
