LOCUS       sequence-423975-        4981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  attB-containing vector backbone, for use in restriction enzyme- or 
            PCR-based cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4981)
  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 4981)
  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: pRL092 pattB_cryaa:Venus_MCS
FEATURES             Location/Qualifiers
     source          1..4981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(255..273)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(294..310)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(307..329)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    318..334
                     /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(342..348,349..366,367..372))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    387..408
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(525..542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(696..1284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(776..795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1455..2246,2247..2315))
                     /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     2078..2097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2316..2420)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2446..2901)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2583..2604)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2795..2814
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3028..3050
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3043..3059
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3066..3085
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3066..3084
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3118..3136)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(3813..3815,3816..3818,3819..4532)
                     /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(3858..3879)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4119..4138)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4466..4487
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4550..4567)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4556..4763
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4795..4811
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4887..4903)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    join(4948..4981,1..36)
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 ttgggctccc cgggcgcgta ctccacctca cccatctggt ccatcatgat gaacgggtcg
       61 aggtggcggt agttgatccc ggcgaacgcg cggcgcaccg ggaagccctc gccctcgaaa
      121 ccgctgggcg cggtggtcac ggtgagcacg ggacgtgcga cggcgtcggc gggtgcggat
      181 acgcggggca gcgtcagcgg gttctcgacg gtcacggcgg gcatgtcgac actagttcta
      241 gccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag
      301 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
      361 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
      421 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
      481 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
      541 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
      601 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
      661 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
      721 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      781 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      841 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      901 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      961 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1021 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1081 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     1141 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     1201 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     1261 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     1321 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     1381 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     1441 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     1501 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     1561 ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc ggctccagat
     1621 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1681 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1741 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     1801 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1861 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1921 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1981 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     2041 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     2101 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     2161 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     2221 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     2281 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     2341 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     2401 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     2461 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2521 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     2581 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     2641 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     2701 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2761 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2821 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2881 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     2941 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     3001 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     3061 aattgtaata cgactcacta tagggcgaat tgggtacgta ccgggcccct agtatgtatg
     3121 taagttaata aaacccattt ttgcggaaag tagataaaaa aaacattttt tttttttact
     3181 gcactggatc tagcggatcc gggaattggg aattcgttaa cagatctgcg gccgcggctc
     3241 gagtaccatt aatagtgtgc attcagtgca ggatgttatg tatttatttt ctgtagtgca
     3301 aactttttca aagaattttc cagtctaagc ttttcagtca agaaaaaaaa aaagcgattg
     3361 attatctgtg atatttaaaa aaattagctc ttaaatctgg tgaaccaagt ggccacaaca
     3421 cattaaatac tgacgttcta cagccatgta gcttcatctg gtctggtttg ttttggcagg
     3481 cacttatttt ccggtgatag ttggccacaa gagtattgtc tgagccattc agtgctagac
     3541 tgtcattctc aggtcagagt ccaccccgct gattctgctg acaacactgt tcccaccatg
     3601 agataatgcc attccagaga gatccatttg taagcccctc tttctgcagc acaggtatat
     3661 aaccaggggt ctgcctccac taaggccggc acacatcatt tggggatctt tgtactgtac
     3721 tagcctctat tagcctccta cttgcgttca gttcacatct tttgttcatc aggtgcaata
     3781 agttacagta aggagttacc aggtctgaca acatggtgag caagggcgag gagctgttca
     3841 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     3901 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca
     3961 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc
     4021 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     4081 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     4141 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     4201 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     4261 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     4321 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     4381 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc gccctgagca
     4441 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     4501 tcactctcgg catggacgag ctgtacaagt aaagcggccc gctgatcagc ctcgactgtg
     4561 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     4621 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     4681 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     4741 gacaatagca ggcatgctgg ggagagatcg ccgccaccgc ggtgggtact cgagcaggaa
     4801 acagctatga ccatgattac gccaagctat atgcatgata tcgctagcca tatggagctc
     4861 ggtgaccagg cctaccggta taattcactg gccgtcgttt tacggtacct ctagagatcc
     4921 actagtgtcg acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc
     4981 c
//
