LOCUS       sequence-7441-ex        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6558)
  AUTHORS   Villefranc JA, Amigo J, Lawson ND
  TITLE     Gateway compatible vectors for analysis of gene function in the 
            zebrafish.
  JOURNAL   Dev Dyn. 2007 Nov . 236(11):3077-87.
  PUBMED    17948311
REFERENCE   2  (bases 1 to 6558)
  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: "Dev Dyn. 
            2007 Nov . 236(11):3077-87."
COMMENT     Sequence Label: 223 pCS EGFP DEST
FEATURES             Location/Qualifiers
     source          1..6558
                     /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"
     regulatory      119..128
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(125..127,128..130,131..841)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(125..127,128..130,131..841)
                     /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(170..191)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(431..450)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     778..799
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    863..987
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1012..1017,1018..1035,1036..1042)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1096..1755
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1162..1181)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2097..2402
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2281..2300
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2443..2567)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    2607..2741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2631..2650)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2685..2704
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2854..2874)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2854..2872)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2893..2909)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2893..2909)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2906..2928)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2917..2933
                     /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(2941..2947,2948..2965,2966..2971))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2986..3007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3124..3141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3295..3883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3375..3394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4054..4845,4846..4914))
                     /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     4677..4696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4915..5019)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5045..5500)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5182..5203)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5394..5413
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5574..6558
                     /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 aggctagcgc taccggtcgc
      121 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      181 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      241 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      301 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      361 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      421 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      481 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
      541 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
      601 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
      661 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
      721 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
      781 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
      841 gtccggactc agatctcgaa tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa
      901 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
      961 taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt
     1021 tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccgtcgag attttcagga
     1081 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     1141 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     1201 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     1261 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg
     1321 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
     1381 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
     1441 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
     1501 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
     1561 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
     1621 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
     1681 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     1741 cagggcgggg cgtaaacgcg tggatccggc ttactaaaag ccagataaca gtatgcgtat
     1801 ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca
     1861 aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt
     1921 tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga
     1981 agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt
     2041 cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct ggtgaaatgc
     2101 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
     2161 gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     2221 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     2281 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     2341 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     2401 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt
     2461 tgtgttttac agcattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata
     2521 tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtggtgat tcgagcctct
     2581 agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg
     2641 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2701 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2761 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc
     2821 caatgcattg ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     2881 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2941 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3001 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3061 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3121 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3181 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3241 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3301 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3361 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3421 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3481 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3541 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3601 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3661 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3721 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3781 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3841 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3901 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3961 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4021 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4081 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4141 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4201 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4261 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4321 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4381 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4441 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4501 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4561 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4621 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4681 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4741 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4801 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4861 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4921 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4981 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5041 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5101 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5161 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5221 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5281 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5341 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5401 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5461 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     5521 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccata
     5581 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
     5641 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
     5701 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ttggcactgt
     5761 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
     5821 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
     5881 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
     5941 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
     6001 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
     6061 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
     6121 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
     6181 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
     6241 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
     6301 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
     6361 tattggaagg acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
     6421 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
     6481 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
     6541 aatgctcgtt tagggaac
//
