LOCUS       sequence-394266-        7371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockdown of Collagen Type 1 alpha 1. Construct has inverted repeats
            to be used in Sleeping Beauty transposon system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7371)
  AUTHORS   Comba A, Faisal SM, Dunn PJ, Argento AE, Hollon TC, Al-Holou WN, 
            Varela ML, Zamler DB, Quass GL, Apostolides PF, Abel C 2nd, Brown 
            CE, Kish PE, Kahana A, Kleer CG, Motsch S, Castro MG, Lowenstein PR
  TITLE     Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an
            actionable target to disrupt tumor progression.
  JOURNAL   Nat Commun. 2022 Jun 24;13(1):3606. doi: 10.1038/s41467-022-31340-1.
  PUBMED    35750880
REFERENCE   2  (bases 1 to 7371)
  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 
            Commun."; date: "2022-06-24"; volume: "13(1):3606. doi"; pages: " 
            10.1038/s41467-022-31340-1"
COMMENT     Sequence Label: pT2/Col1a1/GFP4_Seq 1.1
FEATURES             Location/Qualifiers
     source          1..7371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     LTR             1034..1550
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    1614..1955
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             2022..2438
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2368..2390
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2406..2422
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           2478..2599
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2627..2641
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2669..2795
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(2828..2848)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2833..3162
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3013..3148
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3169..3171,3172..3174,3175..3888)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3214..3235)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3475..3494)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3822..3843
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3961..4475
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4637..4653
                     /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(4661..4667,4668..4685,4686..4691))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4706..4727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5186..5206)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5186..5204)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5225..5241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5225..5241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5249..5265
                     /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(5273..5279,5280..5297,5298..5303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5318..5339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5456..5473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5627..6215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5707..5726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6386..7177,7178..7246))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7009..7028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7247..7351)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg agtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattggagct
      661 cggatcccta tacagttgaa gtcggaagtt tacatacact taagttggag tcattaaaac
      721 tcgtttttca actactccac aaatttcttg ttaacaaaca atagttttgg caagtcagtt
      781 aggacatcta ctttgtgcat gacacaagtc atttttccaa caattgttta cagacagatt
      841 atttcactta taattcactg tatcacaatt ccagtgggtc agaagtttac atacactaag
      901 ttgactgtgc ctttaaacag cttggaaaat tccagaaaat gatgtcatgg ctttagaagc
      961 ttgatctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
     1021 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     1081 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
     1141 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     1201 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
     1261 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     1321 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     1381 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
     1441 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt
     1501 gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc
     1561 accgagattt ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg
     1621 ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt
     1681 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1741 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1801 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1861 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1921 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1981 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     2041 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     2101 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     2161 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     2221 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     2281 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     2341 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     2401 cctcccttta tccagccctc actccttctc taggcgccgg aattagatct ctcgactagg
     2461 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     2521 cttggaaaca cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt
     2581 atattgctgt tgacagtgag cgccctggtg atactggtgt taaatagtga agccacagat
     2641 gtatttaaca ccagtatcac cagggtgcct actgcctcgg aattcaaggg gctactttag
     2701 gagcaattat cttgtttact aaaactgaat accttgctat ctctttgata catttttaca
     2761 aagctgaatt aaaatggtat aaattaaatc acttttttca attcgccagc acagtggtcg
     2821 accctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2881 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2941 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3001 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3061 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3121 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcat ggtgagcaag
     3181 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3241 ggccacaagt tcagcgtgtc tggcgagggc gagggcgatg ccacctacgg caagctgacc
     3301 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3361 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3421 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3481 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3541 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3601 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcg
     3661 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3721 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     3781 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3841 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaatg aattaattaa
     3901 gaattatcaa gcttatcgat aaaataaaag attttattta gtctccagaa aaagggggga
     3961 atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca
     4021 tggaaaatac ataactgaga atagagaagt tcagatcaag gttaggaaca gagagacagc
     4081 agaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag
     4141 aacagatggt ccccagatgc ggtcccgccc tcagcagttt ctagagaacc atcagatgtt
     4201 tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
     4261 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
     4321 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat
     4381 aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct
     4441 gagtgattga ctacccgtca gcgggggtct ttcatgggta acagtttctt gaagttggag
     4501 aacaacattc tgagggtagg agtcgaatat taagtaatcc tgactcaatt agccactgtt
     4561 ttgaatccac atactccaat actcctgaaa tagttcatta tggacagcgc agaaagagct
     4621 ggggagaatt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4681 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4741 cactgcccgc tttccagtcg ggaaacctgt cgtgccagat ccatggactt cactagtgcg
     4801 cgcggccgct ctagatggcc agatctagct tgtggaaggc tactcgaaat gtttgaccca
     4861 agttaaacaa tttaaaggca atgctaccaa atactaattg agtgtatgta aacttctgac
     4921 ccactgggaa tgtgatgaaa gaaataaaag ctgaaatgaa tcattctctc tactattatt
     4981 ctgatatttc acattcttaa aataaagtgg tgatcctaac tgacctaaga cagggaattt
     5041 ttactaggat taaatgtcag gaattgtgaa aaagtgagtt taaatgtatt tggctaaggt
     5101 gtatgtaaac ttccgacttc aactgtatag ggatcctcta gctagagtcg acctcgcggg
     5161 ggggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     5221 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5281 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5341 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5401 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5461 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5521 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5581 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5641 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5701 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5761 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaaccgtg gcgctttctc
     5821 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5881 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5941 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6001 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6061 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6121 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6181 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6241 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6301 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6361 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6421 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6481 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6541 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6601 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6661 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6721 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6781 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6841 gtaacttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6901 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6961 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7021 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7081 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7141 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7201 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7261 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7321 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg c
//
