LOCUS       sequence-321530-        8906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  amplification of Gallus gallus Vcl mutated in Tankyrase Binding 
            Domain II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8906)
  AUTHORS   Vilchez-Larrea, S, Valsecchi W, Fernandez-Villamil S,, Lafon Hughes 
            L
  TITLE     First evidences suggesting a role of a tankyrase-binding motif (TBM)
            of vinculin (VCL) in epithelial cells
  JOURNAL   PeerJ
REFERENCE   2  (bases 1 to 8906)
  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: "PeerJ"
COMMENT     Sequence Label: pET15b/GVcl 1-1066 /*TBM
FEATURES             Location/Qualifiers
     source          1..8906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    122..261
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(187..206)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     347..369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(363..554)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1866..2948)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2910..2929
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2949..3026)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3232..3251
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3335..3354
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3335..3353
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3354..3378
                     /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)."
     RBS             3393..3415
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3434..3451
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3461..3478
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(6741..6759)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6755..6802
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6977..6982,6983..6999,7000..7005))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7032..7050)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7118..7222
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7223..7291,7292..8083)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7441..7460)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8254..8842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8743..8762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
       61 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
      121 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
      181 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
      241 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
      301 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
      361 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
      421 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
      481 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
      541 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
      601 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
      661 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
      721 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
      781 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
      841 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
      901 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
      961 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     1021 ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg
     1081 atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg
     1141 gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg
     1201 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
     1261 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga
     1321 cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
     1381 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga
     1441 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg
     1501 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
     1561 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     1621 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     1681 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     1741 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     1801 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     1861 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1921 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     1981 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     2041 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     2101 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     2161 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     2221 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     2281 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     2341 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     2401 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     2461 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     2521 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     2581 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     2641 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     2701 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     2761 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     2821 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     2881 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     2941 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     3001 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     3061 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     3121 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     3181 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     3241 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     3301 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     3361 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     3421 catgggcagc agccatcatc atcatcatca cagcagcggc ctggtgccgc gcggcagcca
     3481 tatgatgccc gtcttccaca cgcgcaccat cgagagcatc ttggagcccg tggctcagca
     3541 gatctcccac ctggtcatca tgcacgagga gggggaggta gacggcaagg ccatcccgga
     3601 cctcaccgcc cccgtgtcgg ccgtgcaggc cgctgtcagc aacctggtgc gggttggaaa
     3661 agaaactgtg cagacaacag aagaccagat cttgaaaagg gatatgccac cagcattcat
     3721 caaagtagag aatgcctgca ccaagctcgt tcgagcagcc cagatgctgc aagcagatcc
     3781 ttattcagta ccagctcgtg actacctaat tgatggatca agaggcatcc tttctggaac
     3841 atcagactta cttctgacat ttgatgaagc ggaggtccgt aaaatcatcc gtgtctgcaa
     3901 aggaatattg gaatatctga ctgtggcaga agtagtagag actatggagg atttggtgac
     3961 atatacaaag aatctagggc caggaatgac aaagatggcg aaaatgatcg atgagagaca
     4021 acaggaatta actcatcagg aacatagggt tatgctggtg aactccatga atactgtgaa
     4081 ggagctattg cctgtactta tttcagctat gaagatcttt gtaaccacaa aaaacactaa
     4141 aagccaggga atagaagagg ccttgaaaaa tcgcaatttc acagtagaga aaatgagtgc
     4201 tgagataaat gaaataatcc gtgtattaca actcacttcc tgggatgaag atgcctgggc
     4261 cagcaaggac actgaagcca tgaaaagagc tttagcccta atagattcaa agatgaacca
     4321 ggcaaaaggc tggctgagag atccaaacgc acctccaggg gatgctggtg agcaagcaat
     4381 aaggcagatc cttgatgaag ctggaaaagc aggagaattg tgtgcaggca aagaacgcag
     4441 ggagattctg ggaacatgca aaactttggg ccaaatgact gatcaacttg ctgatctccg
     4501 agctagagga cagggtgcta caccgatggc aatgcagaag gcacagcagg tgtcacaagg
     4561 cctggatttg ctcactgcaa aagtggagaa tgcagcccgg aaattggagg ccatgacaaa
     4621 ctctaagcag gctattgcaa agaagattga tgctgctcag aactggcttg cggatcccaa
     4681 cggcggaagt gaaggagaag aacacattcg aggaattatg tctgaagcaa ggaaagttgc
     4741 agaattgtgt gaggagccta aagaaagaga tgatatcctt cgctccttgg gggaaatctc
     4801 tgctctgaca gctaagctgt cagatctgcg acgacatggg aaagtagact ctcctgaggc
     4861 ccgtgcattg gccaagcaaa tagctacatc acttcagaac ttacagtcca aaacaaacag
     4921 agctgtagca aatactaggc cagttaaagc tgctgtccat ttggagggca agattgagca
     4981 agcccagagg tggatagata atcctacagt tgatgatcgg ggagtaggcc aggcagcaat
     5041 tcggggtttg gttgcagaag gtcgtcgtct agccaatgtc atgatgggac cttatcgtca
     5101 ggacctgctt gccaaatgtg accgtgtaga ccagctggct gctcagcttg ctgaccttgc
     5161 agcaagaggg gaaggagagt ctcctcaggc tagggcaatt gctgctcagc ttcaggactc
     5221 cctgaaggat ctcaaagcac ggatgcaaga agcaatgacc caggaggtgt ctgatgtttt
     5281 cagtgacact acaaccccta ttaagttgtt agcagtagca gccactgctc cttctgatac
     5341 tcccaataga gaagaggtgt ttgaagaaag agcagcaaat tttgaaaacc atgctgctag
     5401 actgggagca acagcagaaa aagcagctgc agttggaact gctaataaaa ctactgtgga
     5461 aggcattcag gcaacagtca aatctgcaag ggagcttaca ccacaggtag tatcggctgc
     5521 tcgaatcctc ctgagaaatc ctggaaatca agctgcttat gagcattttg agacaatgaa
     5581 aaaccaatgg attgataatg tagaaaagat gacagggctg gtggatgagg ccatcgatac
     5641 caagtctctg ttggatgcat cagaagaggc tattaagaag gatcttgata aatgcaaagt
     5701 tgcaatggcc aatatgcaac ctcagatgct ggtagctgga gccaccagca ttgctagacg
     5761 agcgaaccgc atcctgcttg tggcaaaacg ggaggttgaa aattcagaag accctaaatt
     5821 cagggaggct gttaaagcag cttctgatga gctgagcaaa accatatcac cgatggtaat
     5881 ggatgctaaa gctgtagcag gaaatatctc tgatcctggt ttgcagaaga gtttcttgga
     5941 ttctggatac aggattctgg gagctgtggc caaagtcaga gaagcctttc agcctcagga
     6001 gccagacttt ccccctcctc ctcctgacct agagcatctc catctgactg atgagcttgc
     6061 tcctccaaaa ccaccacttc cagaaggtga ggttccccca cccagaccac caccccctga
     6121 agaaaaagat gaggagttcc cagagcagaa agcaggagaa gctattaatc agcccatgat
     6181 gatggctgct aggcagttgc atgacgaggc tcggaaatgg tctagcaagg gtaacgacat
     6241 cattgctgct gctaaacgaa tggcgctgct catggctgag atgtcacgcc tggtgcgagg
     6301 aggcagcgga aacaagcgtg ccctcatcca gtgtgcaaaa gatattgcta aggcatcgga
     6361 tgaagtcact cgattggcca aagaggtggc caagcaatgt actgataaac gcattagaac
     6421 aaacctctta caggtctgtg agcgaatccc aaccatcagc acgcagctca aaattctttc
     6481 cacagtgaaa gctaccatgc tgggcaggac taacatcagc gatgaagaat cagaacaggc
     6541 aactgagatg ttggttcata acgcccagaa cctcatgcag tctgtgaagg aaactgtgag
     6601 agaagctgaa gcagcatcca ttaagataag aacagatgcc ggattcactc tgcgctgggt
     6661 cagaaagacc ccatggtatc agtagctcga ggatccggct gctaacaaag cccgaaagga
     6721 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     6781 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     6841 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     6901 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     6961 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     7021 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     7081 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     7141 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     7201 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     7261 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     7321 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     7381 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     7441 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     7501 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     7561 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     7621 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     7681 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     7741 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     7801 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     7861 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     7921 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     7981 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     8041 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     8101 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     8161 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     8221 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     8281 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     8341 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     8401 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     8461 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     8521 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     8581 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     8641 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     8701 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     8761 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     8821 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     8881 ttgctggcct tttgctcaca tgttct
//
