LOCUS       sequence-55346-e        7042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7042)
  AUTHORS   Schmid-Burgk JL, Schmidt T, Kaiser V, Honing K, Hornung V
  TITLE     A ligation-independent cloning technique for high-throughput 
            assembly of transcription activator-like effector genes.
  JOURNAL   Nat Biotechnol. 2012 Dec 16. doi: 10.1038/nbt.2460.
  PUBMED    23242165
REFERENCE   2  (bases 1 to 7042)
  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: "Nat 
            Biotechnol. 2012 Dec 16. doi: 10.1038/nbt.2460."
COMMENT     Sequence Label: TALEN-ID12-C
FEATURES             Location/Qualifiers
     source          1..7042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        913..931
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             980..1045
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1052..1072
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1831..2418
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     protein_bind    complement(2440..2473)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(2594..2612)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2595..2612)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2632..2649)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2638..2862
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2908..3336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2995..3014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3205..3226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3345..3365)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3350..3680
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3531..3666
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3593..3612
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3747..4541
                     /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(3801..3820)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4411..4430
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4715..4836
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4752..4771)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4806..4825
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4885..4901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4885..4901)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4898..4920)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4909..4925
                     /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(4933..4939,4940..4957,4958..4963))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4978..4999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5116..5133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5287..5875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5367..5386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6046..6837,6838..6906))
                     /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     6669..6688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6907..7011)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggaccc
      901 tcgtacagaa gctaatacga ctcactatag ggaaataaga gagaaaagaa gagtaagaag
      961 aaataataga ctcgagatgg actacaagga ccacgacggc gactacaagg accacgacat
     1021 cgactacaag gacgacgacg acaagatggc ccccaagaag aagagaaagg tgggcatcca
     1081 cagaggcgtg cccatggtgg acctgagaac cctgggctac agccagcagc agcaggagaa
     1141 gatcaagccc aaggtgagaa gcaccgtggc ccagcaccac gaggccctgg tgggccacgg
     1201 cttcacccac gcccacatcg tggccctgag ccagcacccc gccgccctgg gcaccgtggc
     1261 cgtgaagtac caggacatga tcgccgccct gcccgaggcc acccacgagg ccatcgtggg
     1321 cgtgggcaag cagtggagcg gcgccagagc cctggaggcc ctgctgaccg tggccggcga
     1381 gctgagaggc ccccccctcc agctggacac cggccagctg ctgaagatcg ccaagagagg
     1441 cggcgtgacc gccgtggagg ccgtgcacgc ctggagaaac gccctgaccg gcgcccccct
     1501 gaatttgaca cccgaccaag gtaccggtgg atccggtggt accggttgct ccctgtcttg
     1561 tgtcaagacc atgggctgac ccccgagcag gtggtggcca tcgccagcca cgacggcggc
     1621 agacccgccc tggagagcat cgtggcccag ctgagcagac ccgaccccgc cctggccgcc
     1681 ctgaccaacg accacctggt ggccctggcc tgcctgggcg gcagacccgc cctggacgcc
     1741 gtgaagaagg gcctgcccca cgcccccgcc ctgatcaaga gaaccaacag aagaatcccc
     1801 gagagaacca gccacagagt ggccggcagc cagctggtga agagcgagct ggaggagaag
     1861 aagagcgagc tgagacacaa gctgaagtac gtgccccacg agtacatcga gctgatcgag
     1921 atcgccagaa acagcaccca ggacagaatc ctggagatga aggtgatgga gttcttcatg
     1981 aaggtgtacg gctacagagg caagcacctg ggcggcagca gaaagcccga cggcgccatc
     2041 tacaccgtgg gcagccccat cgactacggc gtgatcgtgg acaccaaggc ctacagcggc
     2101 ggctacaacc tgcccatcgg ccaggccgac gagatgcaga gatacgtgga ggagaaccag
     2161 accagaaaca agcacatcaa ccccaacgag tggtggaagg tgtaccccag cagcgtgacc
     2221 gagttcaagt tcctgttcgt gagcggccac ttcaagggca actacaaggc ccagctgacc
     2281 agactgaacc acatcaccaa ctgcaacggc gccgtgctga gcgtggagga gctgctgatc
     2341 ggcggcgaga tgatcaaggc cggcaccctg accctggagg aggtgagaag aaagttcaac
     2401 aacggcgaga tcaacttcag aagctctaga tgaactagta taacttcgta tagcatacat
     2461 tatacgaagt tatgctagcg cggccgcgct gccttctgcg gggcttgcct tctggccatg
     2521 cccttcttct ctcccttgac acctgtacct cttggtcttt gaataaagcc tgagtaggaa
     2581 gtgagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg
     2641 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2701 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2761 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2821 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2881 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2941 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3001 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3061 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3121 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3181 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3241 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa
     3301 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3361 agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca
     3421 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3481 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3541 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3601 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3661 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     3721 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3781 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3841 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3901 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3961 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4021 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4081 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4141 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4201 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4261 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4321 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4381 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4441 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4501 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4561 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4621 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4681 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4741 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4801 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4861 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4921 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4981 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5101 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5161 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5221 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5461 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5701 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7021 ccgaaaagtg ccacctgacg tc
//
