LOCUS       sequence-234035-        3936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For making C-terminal sCFP fusions using multisite Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3936)
  AUTHORS   Innes R
  TITLE     multisite Gateway cloning vectors for fluorescent fusions
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3936)
  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            "Unpublished"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: sCFP in pBSDONR P4r-P2
FEATURES             Location/Qualifiers
     source          1..3936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..161
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     153..170
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     154..170
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     180..199
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        180..198
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(253..376)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(379..381,382..384,385..1095)
                     /codon_start=1
                     /product="monomeric optimized variant of ECFP (Kremers et 
                     al., 2006)"
                     /label=SCFP3A
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(424..445)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(685..704)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1032..1053
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1100..1199)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1248..1264)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1248..1264)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1301..1321)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1301..1319)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1340..1356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1340..1356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1353..1375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1364..1380
                     /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(1388..1394,1395..1412,1413..1418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1433..1454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1571..1588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1742..2330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1822..1841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2501..3292,3293..3361))
                     /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     3124..3143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3362..3466)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3493..3922
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3580..3599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3790..3811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 acgcttacaa tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
       61 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
      121 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt
      181 aatacgactc actatagggc gaattgggta ccgggcccct acaggtcact aataccatct
      241 aagtagttga ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
      301 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact
      361 tttctataca aagttgacat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      421 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      481 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      541 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcgcccgc
      601 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      661 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      721 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      781 ggcaacatcc tggggcacaa gctggagtac aactacatca gcgacaacgt ctatatcacc
      841 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
      901 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      961 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     1021 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1081 gacgagctgt acaagtaata cccagctttc ttgtacaaag ttggcattat aagaaagcat
     1141 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     1201 catccagctg cagggcggcc gcgatatcga attcctgcag cccgggggat ccactagttc
     1261 tagagcggcc gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg
     1321 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1381 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1441 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1501 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1561 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1621 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1681 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1741 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1801 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1861 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1921 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1981 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2041 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2101 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2161 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2221 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2281 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2341 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2401 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2461 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     2821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     2941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3361 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3481 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3541 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3781 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3841 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaaa tgagctgatt
     3901 taacaaaaat ttaacgcgaa ttttaacaaa atatta
//
