LOCUS       sequence-160381-        7045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Replaces #61376. QUAS driving GFP with beta crystalline CFP eye 
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7045)
  TITLE     QUAS:GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7045)
  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            "Unpublished"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: Quas:GFPpA;Betacrystallin:CFP
FEATURES             Location/Qualifiers
     source          1..7045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     1061..1077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1097..1117
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1308..1332
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1338..1340,1341..1343,1344..2054)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1338..1340,1341..1343,1344..2054)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2107..2126)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2108..2126)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2131..2265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2327..2351)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3002..3035)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      3188..3197
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3194..3196,3197..3199,3200..3913)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    complement(3942..3962)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(3970..3986)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4002..4136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(4575..4762,4763..4774)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4808..4824)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4808..4824)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4861..4881)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4861..4879)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4900..4916)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4913..4935)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4924..4940
                     /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(4948..4954,4955..4972,4973..4978))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4993..5014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5131..5148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5302..5890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5382..5401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6061..6852,6853..6921))
                     /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     6684..6703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6922..7026)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 aggctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatg ggccctcgag caggaaacag ctatgaccat
     1081 gattacgcca agctatcaac tttgtataga aaagttgggg taatcgctta tcctcggata
     1141 aacaattatc ctcacgggta atcgcttatc cgctcgggta atcgcttatc ctcgggtaat
     1201 cgcttatcct tgcaagggtc gactctagag ggtatataat ggatcccatc gcgtctcagc
     1261 ctcactttga gctcctccac acgaattccc tcgacctcga agacgcgtca agtttgtaca
     1321 aaaaagcagg ctccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1381 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1441 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1501 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1561 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1621 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1681 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1741 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1801 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1861 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1921 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1981 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2041 acgagctgta caagggcggt ggaagatctg ggaattcaag gcctctcgag cctctagatt
     2101 ctgcagccct atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     2161 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2221 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     2281 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaccc agctttcttg
     2341 tacaaagtgg accgtcgagc aattggtgct atcatcatga caaagagaga agaaatcagt
     2401 tattagaaat gagttattaa aactattatg attagaaata tgtttctttc tcttaaacag
     2461 gatttaaggg gaaaatatac agggagcgaa aaattttgac ttcatctgta tatacatata
     2521 ataaataata catcaataaa ttccatctaa atacgctacc atatatatat atatatgtat
     2581 atacacacac acacacacac catctaatat atgtcatgct acattattaa cttcagtatg
     2641 aaatctactc tgctctggag tgtatcagcg agcgactcct ctgctcggat tatcatcacg
     2701 tgacctctga cctgaacaca gacctctctt agccataggt atacagccta tagcatagag
     2761 ctcgggctga gagggaaagt gccgccgtca gggctttcca ttgacagtaa aatgcttacg
     2821 gccttctggg cgatgcgatg accctgcgct tcggcacggc tgaactctga tgacggcgtg
     2881 tttctattgt ccgccgcgcg ccgctgtgct gcccttgcca gcgtataaaa gctgcggcag
     2941 tggtgaggaa ggccagagcg agccagcctt cccagcacag ctgtagttta aacgcgttgc
     3001 cataacttcg tataggatac tttatacgaa gttatctgca tgctaggttg ccgggaacgg
     3061 tgcattggaa cgcgcattcc ccgtgttaat taacaggtaa gtgtcttcct cctgtttcct
     3121 tcccctgcta ttctgctcaa ccttcctatc agaaactgca gtatctgtat ttttgctagg
     3181 ggatcccgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     3241 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3301 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3361 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc
     3421 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3481 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3541 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3601 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga
     3661 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag
     3721 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3781 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     3841 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     3901 gctgtacaag taaagcggcc aaacccgctg atcagcctcg acaactttat tatacatagt
     3961 tgataattca ctggccgtcg ttttacggta ccatcgatga tgatccagac atgataagat
     4021 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     4081 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     4141 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     4201 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagat cagatctgcg
     4261 aagatacggc cacgggtgct cttgatcctg tggctgattt tggactgtgc tgctcgcagc
     4321 tgctgatgaa tcacatactt cctccatttt cttccactga ttgactgtta taatttccct
     4381 aatttccagg tcaaggtgct gtgcattgtg gtaatagatg tgacatgacg tcacttccaa
     4441 aggaccaatg aacatgtctg accaatttca tataatgtga aaacgatttt cataggcaga
     4501 ataaataaca tttaaattaa actgggcatc agcgcaattc aattggtttg gtaatagcaa
     4561 gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg
     4621 taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt
     4681 gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa
     4741 tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac cttcagcaag
     4801 cccagcagat ccactagttc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
     4861 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4921 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4981 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5041 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5101 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5161 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5221 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5281 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5341 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5401 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5461 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5521 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5581 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5641 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5701 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5761 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5821 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5881 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5941 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6001 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6061 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6121 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6181 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6241 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6301 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6361 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6421 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6481 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6541 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6601 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6661 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6721 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6781 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6841 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6901 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6961 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7021 tccgcgcaca tttccccgaa aagtg
//
