LOCUS       sequence-342173-        4301 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 4301)
  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 4301)
  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..4301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..34)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(18..34)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(31..53)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    42..58
                     /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(66..72,73..90,91..96))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    111..132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(249..266)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(420..1008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(500..519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1179..1970,1971..2039))
                     /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     1802..1821
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2040..2144)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2222..2677)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2359..2380)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2571..2590
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2803..2825
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2817..2834
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2818..2834
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2841..2860
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2841..2859
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(2903..3040,3041..3052)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     CDS             3214..4008
                     /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
                     LATRDIAEELXGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3268..3287)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3878..3897
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    join(4009..4020,4021..4208)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        complement(4283..4301)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4284..4301)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
       61 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
      121 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
      181 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
      241 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      301 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      361 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      421 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      481 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      541 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      601 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      661 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      721 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      781 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      841 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
      901 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
      961 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1021 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1081 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     1141 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1201 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1261 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     1321 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1381 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1441 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1501 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     1561 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     1621 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     1681 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     1741 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     1801 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     1861 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     1921 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     1981 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     2041 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2101 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2161 gtgccacctg atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     2221 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2281 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2341 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2401 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2461 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2521 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2581 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2641 acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca
     2701 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     2761 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     2821 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggc ccgacgtatt
     2881 accctgttat ccctacttcc ttaatactca agtacaattt taatggagta cttttttact
     2941 tttactcaag taagattcta gccagatact tttactttta attgagtaaa attttcccta
     3001 agtacttgta ctttcacttg agtaaaattt ttgagtactt tttacacctc tgctcactcg
     3061 tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc
     3121 tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga
     3181 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3241 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3301 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3361 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3421 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3481 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3541 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3601 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3661 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3721 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3781 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3841 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3901 gaagagcttg ncggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3961 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaca gaggtgtaaa
     4021 gtacttgagt aattttactt gattactgta cttaagtatt atttttgggg atttttactt
     4081 tacttgagta caattaaaaa tcaatacttt tacttttact taattacatt tttttagaaa
     4141 aaaaagtact ttttactcct tacaatttta tttacagtca aaaagtactt attttttgga
     4201 gatcacttca ttctattttc cctagggata acagggtaat tatgggagag ctcccaacgc
     4261 gttggatgca tagcttgagt attctatagt gtcacctaaa t
//
