LOCUS       sequence-148112-        3618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""intermediate vector"" for the two-step cloning method (Wang and 
            Szaro, Dev Biol, 2015).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3618)
  AUTHORS   Wang C, Szaro BG
  TITLE     A method for using direct injection of plasmid DNA to study 
            cis-regulatory element activity in F0 Xenopus embryos and tadpoles.
  JOURNAL   Dev Biol. 2015 Feb 1;398(1):11-23. doi: 10.1016/j.ydbio.2014.11.010.
            Epub 2014 Nov 20.
  PUBMED    25448690
REFERENCE   2  (bases 1 to 3618)
  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"; doi: 
            "10.1016/j.ydbio.2014.11.010"; journalName: "Dev Biol"; date: 
            "2015-02-1- 1"; volume: "398"; issue: "1"; pages: "11-23"
COMMENT     Sequence Label: pBluescriptII[attB/Ins1]
FEATURES             Location/Qualifiers
     source          1..3618
                     /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     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        752..770
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     752..769
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1430..1450)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1430..1448)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1469..1485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1469..1485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1482..1504)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1493..1509
                     /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(1517..1523,1524..1541,1542..1547))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1562..1583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1700..1717)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1871..2459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1951..1970)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2630..3421,3422..3490))
                     /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     3253..3272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3491..3595)
                     /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 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgaattg aatttaggtg acactataga agagctatga
      781 cgtcgcggtg cgggtgccag gggcgtgccc ttgacgtcgc atgcacgcgt acgtagacag
      841 actcatatcc gcgggagctc acggggacag ccccccccca aagcccccag ggctgtaatt
      901 gcgttcctct cccgctaggg ggcagcagcg agccgcccag ggctccgctc cggcccgacg
      961 ctccccccgc atccccgagc cggagcccgc agcgtgcggg gacagcccgg cacggggaag
     1021 gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct
     1081 ggggggatac ggggaaaaag ctttatctag atccgcggga gctcacgggg acagcccccc
     1141 cccaaagccc ccagggctgt aattgcgttc ctctcccgct agggggcagc agcgagccgc
     1201 ccggggctcc gctccggccc gacgctcccc ccgcatcccc gagccggagc ccgcagcgtg
     1261 cggggacagc ccggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc
     1321 tgctctttga gcctgcagac acctgggggg atacggggaa aaagctttat cgctctgtcg
     1381 cactagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     1441 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     1501 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     1561 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1621 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1681 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1741 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1801 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1861 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1921 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1981 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2041 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2101 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2161 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2221 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2281 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     2341 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2401 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2461 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2521 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2581 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2641 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2701 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2761 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     2821 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     2881 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     2941 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3001 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3061 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3121 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3181 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3241 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3301 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3361 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3421 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3481 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3541 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3601 ttccccgaaa agtgccac
//
