LOCUS       sequence-65138-e        9017 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 9017)
  AUTHORS   Cohen DM, Chen H, Johnson RP, Choudhury B, Craig SW
  TITLE     Two distinct head-tail interfaces cooperate to suppress activation 
            of vinculin by talin.
  JOURNAL   J Biol Chem. 2005 Apr 29;280(17):17109-17. Epub 2005 Feb 22.
  PUBMED    15728584
REFERENCE   2  (bases 1 to 9017)
  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: "J Biol 
            Chem."; date: "2005-04-29"; volume: "280(17)"; pages: "17109-17. 
            Epub 2005 Feb 22"
COMMENT     Sequence Label: pET15b/EYFP-GgVcl 1-851
FEATURES             Location/Qualifiers
     source          1..9017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2082..2101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2242..2264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3761..4843)
                     /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     4805..4824
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4844..4921)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5127..5146
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5230..5249
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5230..5248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5249..5273
                     /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             5288..5310
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5329..5346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5356..5373
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5377..5379,5380..5382,5383..6093)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5422..5443)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5683..5702)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6030..6051
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(8744..8762)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8758..8805
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8980..8985,8986..9002,9003..9008))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt
     3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     4021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     4141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     4201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     4261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     4321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4861 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     4981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5041 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     5101 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     5161 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     5221 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     5281 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     5341 catcacagca gcggcctggt gccgcgcggc agccatatgg tgagcaaggg cgaggagctg
     5401 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     5461 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     5521 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc
     5581 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     5641 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     5701 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     5761 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     5821 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     5881 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     5941 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg
     6001 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     6061 gggatcactc tcggcatgga cgagctgtac aagtcgaatt ccggcccccc ctgccccgcc
     6121 gccgcccata tgatgcccgt cttccacacg cgcaccatcg agagcatctt ggagcccgtg
     6181 gctcagcaga tctcccacct ggtcatcatg cacgaggagg gggaggtaga cggcaaggcc
     6241 atcccggacc tcaccgcccc cgtgtcggcc gtgcaggccg ctgtcagcaa cctggtgcgg
     6301 gttggaaaag aaactgtgca gacaacagaa gaccagatct tgaaaaggga tatgccacca
     6361 gcattcatca aagtagagaa tgcctgcacc aagctcgttc gagcagccca gatgctgcaa
     6421 gcagatcctt attcagtacc agctcgtgac tacctaattg atggatcaag aggcatcctt
     6481 tctggaacat cagacttact tctgacattt gatgaagcgg aggtccgtaa aatcatccgt
     6541 gtctgcaaag gaatattgga atatctgact gtggcagaag tagtagagac tatggaggat
     6601 ttggtgacat atacaaagaa tctagggcca ggaatgacaa agatggcgaa aatgatcgat
     6661 gagagacaac aggaattaac tcatcaggaa catagggtta tgctggtgaa ctccatgaat
     6721 actgtgaagg agctattgcc tgtacttatt tcagctatga agatctttgt aaccacaaaa
     6781 aacactaaaa gccagggaat agaagaggcc ttgaaaaatc gcaatttcac agtagagaaa
     6841 atgagtgctg agataaatga aataatccgt gtattacaac tcacttcctg ggatgaagat
     6901 gcctgggcca gcaaggacac tgaagccatg aaaagagctt tagccctaat agattcaaag
     6961 atgaaccagg caaaaggctg gctgagagat ccaaacgcac ctccagggga tgctggtgag
     7021 caagcaataa ggcagatcct tgatgaagct ggaaaagcag gagaattgtg tgcaggcaaa
     7081 gaacgcaggg agattctggg aacatgcaaa actttgggcc aaatgactga tcaacttgct
     7141 gatctccgag ctagaggaca gggtgctaca ccgatggcaa tgcagaaggc acagcaggtg
     7201 tcacaaggcc tggatttgct cactgcaaaa gtggagaatg cagcccggaa attggaggcc
     7261 atgacaaact ctaagcaggc tattgcaaag aagattgatg ctgctcagaa ctggcttgcg
     7321 gatcccaacg gcggaagtga aggagaagaa cacattcgag gaattatgtc tgaagcaagg
     7381 aaagttgcag aattgtgtga ggagcctaaa gaaagagatg atatccttcg ctccttgggg
     7441 gaaatctctg ctctgacagc taagctgtca gatctgcgac gacatgggaa aggcgactct
     7501 cctgaggccc gtgcattggc caagcaaata gctacatcac ttcagaactt acagtccaaa
     7561 acaaacagag ctgtagcaaa tactaggcca gttaaagctg ctgtccattt ggagggcaag
     7621 attgagcaag cccagaggtg gatagataat cctacagttg atgatcgggg agtaggccag
     7681 gcagcaattc ggggtttggt tgcagaaggt cgtcgtctag ccaatgtcat gatgggacct
     7741 tatcgtcagg acctgcttgc caaatgtgac cgtgtagacc agctggctgc tcagcttgct
     7801 gaccttgcag caagagggga aggagagtct cctcaggcta gggcaattgc tgctcagctt
     7861 caggactccc tgaaggatct caaagcacgg atgcaagaag caatgaccca ggaggtgtct
     7921 gatgttttca gtgacactac aacccctatt aagttgttag cagtagcagc cactgctcct
     7981 tctgatactc ccaatagaga agaggtgttt gaagaaagag cagcaaattt tgaaaaccat
     8041 gctgctagac tgggagcaac agcagaaaaa gcagctgcag ttggaactgc taataaaact
     8101 actgtggaag gcattcaggc aacagtcaaa tctgcaaggg agcttacacc acaggtagta
     8161 tcggctgctc gaatcctcct gagaaatcct ggaaatcaag ctgcttatga gcattttgag
     8221 acaatgaaaa accaatggat tgataatgta gaaaagatga cagggctggt ggatgaggcc
     8281 atcgatacca agtctctgtt ggatgcatca gaagaggcta ttaagaagga tcttgataaa
     8341 tgcaaagttg caatggccaa tatgcaacct cagatgctgg tagctggagc caccagcatt
     8401 gctagacgag cgaaccgcat cctgcttgtg gcaaaacggg aggttgaaaa ttcagaagac
     8461 cctaaattca gggaggctgt taaagcagct tctgatgagc tgagcaaaac catatcaccg
     8521 atggtaatgg atgctaaagc tgtagcagga aatatctctg atcctggttt gcagaagagt
     8581 ttcttggatt ctggatacag gattctggga gctgtggcca aagtcagaga agcctttcag
     8641 cctcaggagc cagactttcc ccctcctcct cctgacctag agcattaact cgaggatccg
     8701 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     8761 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     8821 atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     8881 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     8941 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa
     9001 gctgtcaaac atgagaa
//
