LOCUS       sequence-425604-        6634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  attB-containing pDEST plasmid, for use in Multisite Gateway 
            reaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6634)
  AUTHORS   Lalonde RL, Wells HH, Kemmler CL, Nieuwenhuize S, Lerma R, Burger A,
            Mosimann C
  TITLE     pIGLET: Safe harbor landing sites for reproducible and efficient 
            transgenesis in zebrafish.
  JOURNAL   Sci Adv. 2024 Jun 7;10(23):eadn6603. doi: 10.1126/sciadv.adn6603. 
            Epub 2024 Jun 5.
  PUBMED    38838146
REFERENCE   2  (bases 1 to 6634)
  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: "Sci Adv."; 
            date: "2024-06-7"; volume: "10(23):eadn6603. doi"; pages: " 
            10.1126/sciadv.adn6603. Epub 2024 Jun 5"
COMMENT     Sequence Label: pRL055 pDESTattB_cryaa:Venus
FEATURES             Location/Qualifiers
     source          1..6634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1370..1372,1373..1375,1376..2089)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1415..1436)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1676..1695)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2023..2044
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2107..2124)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2113..2320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2352..2368
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2388..2511
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2552..2857)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2654..2673)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3177..3857)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     3772..3791
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3911..3917,3918..3935,3936..3941))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3966..4089)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4097..4113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4158..4227
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(4446..4464)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4485..4501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4498..4520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4509..4525
                     /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(4533..4539,4540..4557,4558..4563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4578..4599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4716..4733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4887..5475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4967..4986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5646..6437,6438..6506))
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6507..6611)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatctag cggatccggg aattgggaat tcgttaacag
      781 atctgcggcc gcggctcgag taccattaat agtgtgcatt cagtgcagga tgttatgtat
      841 ttattttctg tagtgcaaac tttttcaaag aattttccag tctaagcttt tcagtcaaga
      901 aaaaaaaaaa gcgattgatt atctgtgata tttaaaaaaa ttagctctta aatctggtga
      961 accaagtggc cacaacacat taaatactga cgttctacag ccatgtagct tcatctggtc
     1021 tggtttgttt tggcaggcac ttattttccg gtgatagttg gccacaagag tattgtctga
     1081 gccattcagt gctagactgt cattctcagg tcagagtcca ccccgctgat tctgctgaca
     1141 acactgttcc caccatgaga taatgccatt ccagagagat ccatttgtaa gcccctcttt
     1201 ctgcagcaca ggtatataac caggggtctg cctccactaa ggccggcaca catcatttgg
     1261 ggatctttgt actgtactag cctctattag cctcctactt gcgttcagtt cacatctttt
     1321 gttcatcagg tgcaataagt tacagtaagg agttaccagg tctgacaaca tggtgagcaa
     1381 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1441 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1501 cctgaagctg atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1561 cctgggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     1621 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1681 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1741 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1801 caactacaac agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     1861 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     1921 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
     1981 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2041 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggcccgct
     2101 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2161 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2221 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2281 agggggagga ttgggaagac aatagcaggc atgctgggga gagatcgccg ccaccgcggt
     2341 gggtactcga gcaggaaaca gctatgacca tgattacgcc aagctatcaa ctttgtatag
     2401 aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca
     2461 taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat ggtcgacctg
     2521 cagactggct gtgtataagg gagcctgaca tttatattcc ccagaacatc aggttaatgg
     2581 cgtttttgat gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga
     2641 ccggcacact ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca
     2701 ccgggtaaag ttcacggggg actttatctg acagcagacg tgcactggcc agggggatca
     2761 ccatccgtcg cccgggcgtg tcaataatat cactctgtac atccacaaac agacgataac
     2821 ggctctctct tttataggtg taaaccttaa actgcatttc accagcccct gttctcgtcg
     2881 gcaaaggagc cgttcatttc aataaaccgg gcgacctcag ccatcccttc ctgattttcc
     2941 gctttccagc gttcggcacg cagacgacgg gcttcattct gcatggttgt gcttaccgaa
     3001 ccggagatat tgacatcata tatgccttga gcaactgata gctgtcgctg tcaactgtca
     3061 ctgtaatacg ctgcttcata gcatacctct ttttgacata cttcgggtat acatatcagt
     3121 atatattctt ataccgcaaa aatcagcgcg caaatacgca tactgttatc tggcttttag
     3181 taagccggat cctctagatt acgccccgcc tgccactcat cgcagtactg ttgtaattca
     3241 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     3301 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     3361 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     3421 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     3481 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     3541 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     3601 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     3661 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     3721 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     3781 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     3841 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgacgg atcctaactc
     3901 aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt gcctaatgcg
     3961 gccgccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     4021 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca actttattat
     4081 acatagttga taattcactg gccgtcgttt tacggtacct ctagagatcc actagtgtcg
     4141 acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc cttgggctcc
     4201 ccgggcgcgt actccacctc acccatctgg tccatcatga tgaacgggtc gaggtggcgg
     4261 tagttgatcc cggcgaacgc gcggcgcacc gggaagccct cgccctcgaa accgctgggc
     4321 gcggtggtca cggtgagcac gggacgtgcg acggcgtcgg cgggtgcgga tacgcggggc
     4381 agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga cactagttct agccagcttt
     4441 tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct
     4501 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4561 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4621 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4681 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6601 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
