LOCUS       sequence-234042-        3927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For making C-terminal mOrange fusions using multisite Gateway 
            cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3927)
  AUTHORS   Innes R
  TITLE     multisite Gateway cloning vectors for fluorescent fusions
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3927)
  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            "Unpublished"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: mOrange in pBSDONR P4r-P2
FEATURES             Location/Qualifiers
     source          1..3927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..110
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     102..119
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     103..119
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     129..148
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(202..325)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             328..1038
                     /codon_start=1
                     /product="orange monomeric derivative of DsRed fluorescent 
                     protein (Shanet et al., 2004)"
                     /label=mOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     741..760
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1040..1139)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1188..1204)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1188..1204)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1241..1261)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1241..1259)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1280..1296)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1280..1296)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1293..1315)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1304..1320
                     /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(1328..1334,1335..1352,1353..1358))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1373..1394
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1511..1528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1682..2270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1762..1781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2441..3232,3233..3301))
                     /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     3064..3083
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3302..3406)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3433..3889
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3730..3751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
       61 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
      121 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggccccta caggtcacta
      181 ataccatcta agtagttgat tcatagtgac tggatatgtt gtgttttaca gtattatgta
      241 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct
      301 cgttcaactt ttctatacaa agttgacatg gtgagcaagg gcgaggagaa taacatggcc
      361 atcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
      421 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gctttcagac cgctaagctg
      481 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcacc
      541 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt caagctgtcc
      601 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
      661 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
      721 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
      781 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagat gaggctgaag
      841 ctgaaggacg gcggccacta cacctccgag gtcaagacca cctacaaggc caagaagccc
      901 gtgcagctgc ccggcgccta catcgtcggc atcaagttgg acatcacctc ccacaacgag
      961 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1021 gacgagctgt acaagtaata cccagctttc ttgtacaaag ttggcattat aagaaagcat
     1081 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     1141 catccagctg cagggcggcc gcgatatcga attcctgcag cccgggggat ccactagttc
     1201 tagagcggcc gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg
     1261 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     1321 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     1381 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1441 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1501 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1561 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1621 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1681 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1741 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1801 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1861 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1921 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1981 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2041 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2101 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2161 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2221 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2281 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2341 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2401 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2461 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2521 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2581 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2641 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2701 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     2761 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2821 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     2881 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     2941 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3001 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3061 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3121 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3181 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3241 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3301 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3361 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3421 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3481 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3721 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3781 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaaa tgagctgatt
     3841 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat
     3901 tcaggctgcg caactgttgg gaagggc
//
