LOCUS       sequence-146800-        4915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of wild-type or mutant cDNA fragments 
            (mutation reporter) or complete cDNAs in frame with superfolderGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4915)
  TITLE     pCS2+MCS-P2A-sfGFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4915)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCS2+MCS-P2A-sfGFP
FEATURES             Location/Qualifiers
     source          1..4915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             155..211
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(218..220,221..934)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             218..931
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(248..276)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     871..891
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    964..1098
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(988..1007)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1042..1061
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1211..1231)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1211..1229)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1250..1266)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1250..1266)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1263..1285)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1274..1290
                     /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(1298..1304,1305..1322,1323..1328))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1343..1364
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1481..1498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1652..2240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1732..1751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2411..3202,3203..3271))
                     /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     3034..3053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3272..3376)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3402..3857)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3539..3560)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3751..3770
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3931..4915
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggcctctcg agcctctaga
      121 ttaattaagc gcatgcggcg cgccaggaag cggagctact aacttcagcc tgctgaagca
      181 ggctggagac gtggaggaga accctggacc tggatccatg agcaaaggag aagaactttt
      241 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc
      301 tgtccgtgga gagggtgaag gtgatgctac aaacggaaaa ctcaccctta aatttatttg
      361 cactactgga aaactacctg ttccgtggcc aacacttgtc actactctga cctatggtgt
      421 tcaatgcttt tcccgttatc cggatcacat gaaacggcat gactttttca agagtgccat
      481 gcccgaaggt tatgtacagg aacgcactat atctttcaaa gatgacggga cctacaagac
      541 gcgtgctgaa gtcaagtttg aaggtgatac ccttgttaat cgtatcgagt taaagggtat
      601 tgattttaaa gaagatggaa acattcttgg acacaaactc gagtacaact ttaactcaca
      661 caatgtatac atcacggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattcg
      721 ccacaacgtt gaagatggtt ccgttcaact agcagaccat tatcaacaaa atactccaat
      781 tggcgatggc cctgtccttt taccagacaa ccattacctg tcgacacaat ctgtcctgtc
      841 gaaagatccc aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg
      901 gattacacat ggcatggatg agctctacaa ataatctaga actatagtga gtcgtattac
      961 gtagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     1021 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     1081 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     1141 gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtaccca
     1201 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     1261 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc gggagcataa
     1321 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1381 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1441 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1501 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1561 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1621 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1681 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1741 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1801 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1861 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1921 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1981 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2041 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     2101 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2161 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2221 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2281 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2341 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2401 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2461 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2521 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2581 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2641 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2701 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2761 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     2821 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     2881 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2941 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3001 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3061 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3121 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3181 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3241 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3301 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3361 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     3421 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     3481 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     3541 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     3601 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     3661 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3721 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3781 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3841 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     3901 ggtgcgggcc tcttcgctat tacgccagtc gaccatagcc aattcaatat ggcgtatatg
     3961 gactcatgcc aattcaatat ggtggatctg gacctgtgcc aattcaatat ggcgtatatg
     4021 gactcgtgcc aattcaatat ggtggatctg gaccccagcc aattcaatat ggcggacttg
     4081 gcaccatgcc aattcaatat ggcggacttg gcactgtgcc aactggggag gggtctactt
     4141 ggcacggtgc caagtttgag gaggggtctt ggccctgtgc caagtccgcc atattgaatt
     4201 ggcatggtgc caataatggc ggccatattg gctatatgcc aggatcaata tataggcaat
     4261 atccaatatg gccctatgcc aatatggcta ttggccaggt tcaatactat gtattggccc
     4321 tatgccatat agtattccat atatgggttt tcctattgac gtagatagcc cctcccaatg
     4381 ggcggtccca tataccatat atggggcttc ctaataccgc ccatagccac tcccccattg
     4441 acgtcaatgg tctctatata tggtctttcc tattgacgtc atatgggcgg tcctattgac
     4501 gtatatggcg cctcccccat tgacgtcaat tacggtaaat ggcccgcctg gctcaatgcc
     4561 cattgacgtc aataggacca cccaccattg acgtcaatgg gatggctcat tgcccattca
     4621 tatccgttct cacgccccct attgacgtca atgacggtaa atggcccact tggcagtaca
     4681 tcaatatcta ttaatagtaa cttggcaagt acattactat tggaaggacg ccagggtaca
     4741 ttggcagtac tcccattgac gtcaatggcg gtaaatggcc cgcgatggct gccaagtaca
     4801 tccccattga cgtcaatggg gaggggcaat gacgcaaatg ggcgttccat tgacgtaaat
     4861 gggcggtagg cgtgcctaat gggaggtcta tataagcaat gctcgtttag ggaac
//
