LOCUS       sequence-348336-        4297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for generating PCR templates with Tol2 repeats and Isce sites for 
            BAC recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4297)
  AUTHORS   Shih YH, Portman D, Idrizi F, Grosse A, Lawson ND
  TITLE     Integrated molecular analysis identifies a conserved pericyte gene 
            signature in zebrafish.
  JOURNAL   Development. 2021 Nov 9. pii: 273393. doi: 10.1242/dev.200189.
  PUBMED    34751773
REFERENCE   2  (bases 1 to 4297)
  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: 
            "Development. 2021 Nov 9. pii: 273393. doi: 10.1242/dev.200189."
COMMENT     Sequence Label: pGEM-Isce-Tol2-Tn5-neo
FEATURES             Location/Qualifiers
     source          1..4297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(6..24)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7..24)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(42..58)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(42..58)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(55..77)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    66..82
                     /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(90..96,97..114,115..120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    135..156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(273..290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(444..1032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(524..543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1203..1994,1995..2063))
                     /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     1826..1845
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2064..2168)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2246..2701)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2383..2404)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2595..2614
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2827..2849
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2841..2858
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2842..2858
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2865..2884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2865..2883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(2927..3064,3065..3076)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     CDS             3234..4028
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3288..3307)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3898..3917
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    join(4029..4040,4041..4228)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg
       61 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
      121 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
      181 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
      241 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
      301 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
      361 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
      421 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
      481 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      541 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      601 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
      661 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
      721 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      781 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      841 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      901 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      961 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1021 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     1081 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     1141 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     1201 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     1261 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     1321 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     1381 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     1441 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     1501 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     1561 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     1621 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     1681 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     1741 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1801 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1861 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1921 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1981 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     2041 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     2101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     2161 gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg
     2221 cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg
     2281 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2341 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2401 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2461 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     2521 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2581 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     2641 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     2701 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     2761 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     2821 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     2881 agggcgaatt gggcccgacg tattaccctg ttatccctac ttccttaata ctcaagtaca
     2941 attttaatgg agtacttttt tacttttact caagtaagat tctagccaga tacttttact
     3001 tttaattgag taaaattttc cctaagtact tgtactttca cttgagtaaa atttttgagt
     3061 actttttaca cctctgctcg tggacagcaa gcgaaccgga attgccagct ggggcgccct
     3121 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct
     3181 gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3241 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3301 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3361 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3421 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3481 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3541 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3601 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3661 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3721 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3781 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3841 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3901 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3961 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4021 tcttctgaca gaggtgtaaa gtacttgagt aattttactt gattactgta cttaagtatt
     4081 atttttgggg atttttactt tacttgagta caattaaaaa tcaatacttt tacttttact
     4141 taattacatt tttttagaaa aaaaagtact ttttactcct tacaatttta tttacagtca
     4201 aaaagtactt attttttgga gatcacttca ttctattttc cctagggata acagggtaat
     4261 tatgggagag ctcccaacgc gttggatgca tagcttg
//
